Pectin: Technological and Physiological Properties [1st ed.] 9783030534202, 9783030534219

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Vassilis Kontogiorgos  Editor

Pectin: Technological and Physiological Properties

PectinTechnological and Physiological Properties

Vassilis Kontogiorgos Editor

Pectin: Technological and Physiological Properties

Editor Vassilis Kontogiorgos School of Agriculture and Food Sciences The University of Queensland Brisbane, QLD, Australia

ISBN 978-3-030-53420-2    ISBN 978-3-030-53421-9 (eBook) https://doi.org/10.1007/978-3-030-53421-9 © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Polysaccharides are routinely used in food and pharmaceutical industries as they show a broad range of functional properties that are employed to create structures with reproducible physical properties. In recent years, there is a need to create advanced formulations beyond traditional food applications that, for example, delay lipid digestion or deliver bioactives in the gastrointestinal tract at the site of interest. In addition, another area of focus is the identification of bioactive polysaccharides that exhibit physiological characteristics that may improve health. The need to understand how to form and stabilise structures for these applications has boosted research on the fundamental chemical, physical and physiological properties of polysaccharides. Pectin is an ideal macromolecule for such applications, as it is highly tunable because of its complex chain architecture, type and amount of substituent groups, charge and charge density and, in certain cases, the presence of protein. Achieving these research objectives, however, is not trivial, and strong foundations are always needed. The present book was written with the goal to form the springboard for new research ventures using pectin as the primary biological material, as it covers the entire spectrum of its technological and physiological properties. The book aims to have an educational approach that can be used as a supplementary textbook in relevant university courses that deal, for example, with food chemistry, food physical chemistry, digestion and metabolism, functional biomaterials or biodegradable polymers. The chapters provide all the classic information with a measured, in-­ depth approach instigating curiosity for further reading. The book also deals with current topics such as emulsification and film-forming properties of pectin as well as its gastrointestinal fate and bioactivity. The targeted audience of the book are undergraduate and postgraduate students, and researchers in academia or industry with no previous experience on pectin who would like a short but at the same time a thorough introduction in  all aspects of

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pectin properties. The ambition is that this should be the first book that someone would reach to learn about pectin and I hope that readers find it a useful addition to their reference book collection. Brisbane, QLD, Australia  Vassilis Kontogiorgos

Contents

1 Biosynthesis, Localisation, and Function of Pectins in Plants ������������    1 Yang Yang and Charles T. Anderson 2 Pectin Structure����������������������������������������������������������������������������������������   17 David Ropartz and Marie-Christine Ralet 3 Pectin Degrading Enzymes����������������������������������������������������������������������   37 Estelle Bonnin and Jérôme Pelloux 4 Isolation and Characterisation of Pectin ����������������������������������������������   61 Gordon A. Morris and Hana A. S. Binhamad 5 Emulsification Properties of Pectin��������������������������������������������������������   83 Katerina Alba and Vassilis Kontogiorgos 6 Edible Films and Coatings with Pectin��������������������������������������������������   99 Athina Lazaridou and Costas G. Biliaderis 7 Pectin Gelation and Its Assembly into Functional Materials��������������  125 M. A. K. Williams 8 Digestion and Metabolism of Pectin ������������������������������������������������������  149 Kathrin Haider and Peter Wilde 9 Pectin Bioactivity��������������������������������������������������������������������������������������  165 Huihuang Ding and Steve W. Cui 10 Pectin as Drug-Release Vehicle ��������������������������������������������������������������  189 Ali Assifaoui and Odile Chambin

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Contributors

K.  Alba  Department of Biological Sciences, University of Huddersfield, Huddersfield, UK C. T. Anderson  Department of Biology, The Pennsylvania State University, State College, PA, USA A. Assifaoui  School of Pharmacy, Université de Bourgogne Franche Comté, UMR PAM, PCAV team, Dijon, France C.  G.  Biliaderis  Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece H. A. S. Binhamad  Department of Chemical Sciences, University of Huddersfield, Huddersfield, UK E.  Bonnin  INRAE, Nantes, France

UR

BIA

Biopolymères

Interactions

Assemblages,

O. Chambin  School of Pharmacy, Université de Bourgogne Franche Comté, UMR PAM, PCAV team, Dijon, France S. W. Cui  Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada H.  Ding  Guelph Research and Development Centre, Agriculture and Agri-Food Canada, Guelph, ON, Canada K. Haider  Quadram Institute Bioscience, Norwich Research Park, Norwich, UK V.  Kontogiorgos  School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD, Australia A. Lazaridou  Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece G.  A.  Morris  Department of Chemical Sciences, University of Huddersfield, Huddersfield, UK ix

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Contributors

J. Pelloux  BIOPI Biologie des Plantes et Innovation SFR Condorcet, Université de Picardie, Amiens, France M. C. Ralet  INRAE, Biopolymères Interactions Assemblages, Nantes, France D. Ropartz  INRAE, Biopolymères Interactions Assemblages, Nantes, France P. Wilde  Quadram Institute Bioscience, Norwich Research Park, Norwich, UK M.  A.  K.  Williams  School of Fundamental Sciences, Massey University, Palmerston North, New Zealand The MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand The Riddet Institute, Massey University, Palmerston North, New Zealand Y. Yang  Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, China

About the Editor

Vassilis Kontogiorgos  is a food scientist by training having received all his degrees in Food Science from the Aristotle University of Thessaloniki, Greece (B.Sc. and M.Sc.) and University of Guelph, Canada (Ph.D.). He has worked as an NSERC research fellow at the Agriculture and Agri-Food Canada (Canada) and as faculty member at the Department of Biological Sciences, University of Huddersfield (UK) before joining the School of Agriculture and Food Sciences at the University of Queensland (Australia). His research interests are focused on the area of polysaccharide isolation and characterisation with particular interest on pectin extraction and functionality. He is also working on the physical chemistry of colloidal systems that are fabricated with pectin and other polysaccharides. He is an associate editor of Food Hydrocolloids and Food Biophysics.

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Chapter 1

Biosynthesis, Localisation, and Function of Pectins in Plants Yang Yang and Charles T. Anderson

1.1  Pectin Biosynthesis Pectins are a class of matrix polysaccharides in the cell wall that contains three major domains: homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) (see Chap. 2). Galacturonic acid (GalA) monomers form the backbones of HG and RG-II domains, whereas rhamnose-GalA dimers form the backbone of RG-I. Both types of RG can be linked to HG, and HG and RG-I can also be covalently attached to an arabinogalactan protein (Fig. 1.1) (Atmodjo et al. 2013; Tan et al. 2013). Pectic polysaccharides are synthesised in the Golgi apparatus, with related glycoproteins simultaneously synthesised in different Golgi cisternae (Moore et  al. 1991) (Fig. 1.1). Newly synthesised pectins are packaged into vesicles and delivered to the cell wall (Moore et al. 1991). Approximately 67 transferases, including glycosyltransferases, methyltransferases, and acetyltransferases, are predicted to function in pectin biosynthesis. Glycosyltransferases function in synthesising pectin backbones and adding side chains, whereas methyltransferases and acetyltransferases catalyse pectin methyl-esterification and acetylation, respectively (Mohnen 2008). In addition, the synthesis and correct localisation of nucleotide sugars, which are the substrates for pectin synthesis, require the action of other enzymes, including interconversion enzymes, sugar kinases, UDP-sugar pyrophosphorylases, and nucleotide sugar transporters (Atmodjo et al. 2013). HG, the most abundant domain of pectins, is a linear polymer of galacturonic acid (GalA), and is partially modified with methyl groups and acetyl groups at the Y. Yang Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou, China C. T. Anderson (*) Department of Biology, The Pennsylvania State University, University Park, PA, USA e-mail: [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_1

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Fig. 1.1  Pectin synthesis and domain arrangement. Pectins are synthesised in the Golgi apparatus and secreted to the cell wall. The inset box shows current hypotheses about pectin synthesis: in the consecutive glycosyltransferase model, UDP-GalA or UDP-Rha are consecutively added to the non-reducing end of pectin chains by glycosyltransferases; in the domain synthesis model, pectin domains are elongated separately by glycosyltransferases, then linked with other domains. In the cell wall, pectin domains are arranged in at least three different covalently linked configurations. Figure made with BioRender

C-6 and O-2/O-3 positions, respectively. Galacturonosyltransferases (GAUTs) transfer UDP-GalA onto the non-reducing end of oligogalacturonide, resulting in HG chain elongation (Scheller et al. 1999). In Arabidopsis thaliana, the GAUT and GAUT-LIKE gene families contain 15 and 10 genes, respectively, some of which have been demonstrated to produce Golgi-localised proteins (Kong et  al. 2011; Sterling et  al. 2001). GAUT1 has UDP-GalA transferase activity, and GAUT7 is required for anchoring GAUT1 to the Golgi by forming a GAUT1:GAUT7 complex (Sterling et al. 2006; Atmodjo et al. 2011). GAUT11 also shows transferase activity for HG elongation, which is essential for synthesising the HG regions in RG-I domain (Voiniciuc et  al. 2018), and GAUT4 has UDP-GalA transferase activity when transiently expressed (Biswal et al. 2018). HG undergoes methylesterification and acetylation before delivering to the wall. In A. thaliana, increased methyltransferase activities are detected from the isolated microsomal fractions of COTTON GOLGI-RELATED2 (CGR2) and CGR3 overexpression lines, respectively, indicating that these two genes encode putative methyltransferases (Kim et  al. 2015). Mutations in another putative pectin methyltransferase gene, QUASIMODO2/ TUMOROUS SHOOT DEVELOPMENT2, causes dramatic reduction in HG content (Mouille et  al. 2007). In addition, the gene product of POWDERY MILDEW

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RESISTANT 5, which encodes an acetyltransferase, transfers acetyl-CoA to pectin fragments as an acetyltransferase, and contributes to plant resistance to fungal disease (Chiniquy et al. 2019). In contrast to HG, the RG-I backbone is formed by the repeating disaccharide of rhamnose-GalA (Mohnen 2008). Rhamnosyltransferases add rhamnose residues to the non-reducing end of RG-I oligosaccharides, and presumably, a galacturonosyltransferase alternates with the rhamnosyltransferase adds GalA residues to the RG-I backbone (Takenaka et al. 2018). A RG-I:rhamnosyltransferase (RRT), RRT1, has been characterised from A. thaliana with enzyme activity determined in vitro (Takenaka et al. 2018). The rhamnose residues of RG-I can be modified with side chains containing galactans and/or arabinans. RG-I galactosyltransferase and arabinosyltransferase activities are detected in microsomal fractions from suspension cultured cells and plant tissues (Geshi et al. 2002; Nunan and Scheller 2003). ARABINAN DEFICIENT 1 and 2 encode putative arabinosyltransferases in A. thaliana, and mutation of these genes leads to decreased arabinan content in the cell wall (Harholt et al. 2012). RG-II shares the same backbone structure with HG, but has complex side chains. Xylosyltransferase activity has been determined for Arabidopsis rhamnogalacturonan specific xylosyltransferas E (RGXT) 1, 2, and 3, which link xylose onto the fucose residues in the side chains of RG-II (Egelund et al. 2006, 2008). Up to now, several genes encoding putative RG-II synthesis enzymes have been functionally characterised in terms of their effects on pectin content and plant development (Funakawa and Miwa 2015). However, it remains unclear whether those genes encode bona fide enzymes and how most RG-II backbone and side chain residues are linked to the polymer. Based on current studies, two models are proposed for pectin synthesis (Fig. 1.1): the consecutive glycosyltransferase model and the domain synthesis model (Atmodjo et  al. 2013). In the consecutive glycosyltransferase model, nucleotide-­ sugar residues are consecutively added to the non-reducing end of growing backbones and side chains of pectin. Based on the mechanism of the GAUT1:GAUT7 complex in HG synthesis, this model has been updated into a two-phase model whose synthesis efficiency depends on chain length (Amos et al. 2018). During de novo synthesis and short-chain acceptor elongation, pectin synthesis undergoes a slow phase, which converts to a rapid phase when the chain acceptor reaches 11 residues in length. In the domain synthesis model, HG, RG-I, and RG-II domains are elongated separately by adding nucleotide sugars onto their growing chains, and oligosaccharide domains are then linked to other domains to form the final pectin structure (Atmodjo et al. 2013). However, the identity of any domain-linking proteins, or “pectin ligases,” is unknown.

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1.2  Localisation of Pectins in Plants Pectins are deeply conserved from the Charophyte algal relatives of plants to angiosperms, and their abundance varies among different plant species. Although cell wall structure in non-vascular plants differs from that in vascular plants, pectins can still be detected in cell wall residues of Bryophytes and Charophytes (Popper and Fry 2003). For instance, in Penium margaritaceum, a unicellular Charophyte alga, HG is a major wall component and localises to the outer layer of the cell wall (Domozych 2014). Pectins are enriched in the primary walls of eudicots and non-­grass monocots, making up 20–35% of the dry mass of the cell wall. However, pectins are reported to constitute only 5% and 0.1% of the primary cell walls of grasses and the secondary cell walls of angiosperms, respectively (Vogel 2008). The compositions of the primary cell walls in gymnosperms are similar to those of eudicots, in which GalA is the most abundant uronic acid (Popper and Fry 2004). In early-diverging vascular plants such as lycophytes, the amount of RG-II in the primary cell wall is comparable with that in angiosperms, but higher than in bryophytes. This increase in RG-II content corresponds with the evolution of lignified secondary walls in vascular plants (Matsunaga et al. 2004). The plant cell wall is a unique structure, and about 90% of its dry mass consists of pectins, cellulose, hemicellulose (Albersheim et al. 1996). Prevailing cell wall models indicate that pectins form a gel-like matrix (Fig. 1.2a), with cellulose microfibrils and hemicellulose embedded in this matrix (Lampugnani et  al. 2018). However, pectins exist in close proximity with both hemicelluloses and cellulose, and covalent crosslinks between pectins and hemicellulose have been detected (Broxterman and Schols 2018; Fry 2018). Pectins show dynamic patterns in the walls during cell growth. HG has recently been reported to assemble into nanofilament structures, facilitating anisotropic cell growth (Haas et al. 2020). Putative metabolic pulse-labelling of RG-I also shows reorientation from a diffuse pattern into a fibrillar pattern along with cell elongation (Anderson et  al. 2012) (Fig.  1.2a, b). During cytokinesis, pectins are one of the earliest wall components delivered to the growing cell plate (Samuels et al. 1995). Pectins are enriched in the middle lamella that lies between the primary walls of adjacent cells and function in cell adhesion (Daher and Braybrook 2015) (Fig. 1.2c). In wood cell walls, pectins are irregularly located in the middle lamella, which corresponds to the location where lignin first appears during secondary xylem development (Wi et  al. 2005). They also localise to the boundaries of tip-growing and anisotropically growing cells (Fig. 1.2d, e). Pectin content varies in different tissues, and pectins are enriched in fruits, seed coats, and root caps (Fig. 1.2f–h). In kiwifruit, tomato, and strawberry fruits, pectins comprise over 45% of the dry mass of the cell wall, and relative pectin content increases along with fruit ripening (Redgwell et al. 1997). Therefore, fruits are major sources for extractable pectins (see Chap. 4). In addition, pectins are the dominant component of mucilage, where mainly consist of RG-I and a small proportion of HG (Francoz et al. 2015). During

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Fig. 1.2  Pectin localisation in the cell wall and distribution in cells and tissues. Pectin arrangements in the cell wall are shown in (a) and (b): a diffuse pattern (a) and a fibrillar pattern (b). Pectin distributions at the cellular level are shown in (c–e). In the middle lamella (c), methylesterified pectins (green) and demethylesterified pectins (magenta) are enriched at boundaries between adjacent cells and corner junctions, respectively. In the pollen tube wall (d), methylesterified pectins and demethylesterified pectins are asymmetrically located at apical and longitudinal walls, respectively. During lobe formation in pavement cells (e), demethylesterified pectins are enriched at neck regions of periclinal walls, and are less abundant at lobe regions of periclinal walls. Coupled with wall expansion, pectins within anticlinal walls undergo demethylesterification at walls adjacent to new lobe regions. At the tissue level, pectins are enriched in softening fruits (f). Mucilage released at the surface of root cap (g) and seed coat (h) is high in demethylesterifed pectins, which shows a red colour with Ruthenium red staining. PM, plasma membrane. Figure made with BioRender

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seed germination and root growth, mucilage is extruded at the surface of seed coat and root cap, facilitating seed imbibition and root protection (Iijima et  al. 2004; Western et al. 2000).

1.3  Pectin Functions in Plants In plants, the cell wall not only serves as a stiff structure to withstand turgor pressure and external stresses, but also possesses the extensibility to facilitate cell growth and constrain cell shape (Cosgrove 2018). Pectin structure, status and localisation are important influencers of wall structure and mechanics, which modulate cell dynamics and further regulate plant development. Pectin modifications in particular are central in modulating wall mechanics. In the cell wall, pectins undergo a series of modifications. With the action of pectin methylesterases (PMEs) and pectin acetylesterases (PAEs), methyl and acetyl groups are removed from HG, respectively (Bonnin et al. 2008; Bordenave 1996). Demethylesterified HG can either be crosslinked by calcium, resulting in wall stiffening, or be degraded by enzymes like polygalacturonases (PGs) and pectate lyases (PLs), resulting in wall softening (Hocq et  al. 2017). The activities of PMEs and PGs are negatively regulated by pectin methylesterase inhibitors (PMEIs) and polygalacturonase inhibitors (PGIPs), respectively (Sénéchal et  al. 2014). For more information about HG-modifying enzymes, see Chap. 3. Below, we summarise the functions of pectins in cell dynamics and how these processes influence plant development and stress responses (Fig. 1.3). The middle lamella functions as a glue for adhering adjacent cells together. The absence of cellulose and hemicellulose in the middle lamella makes pectins strong candidates for mediating cell adhesion and separation (Jarvis et  al. 2003). Cell adhesion is mainly controlled at thickened corner junctions, which are strengthened by demethylesterified pectin with calcium-crosslinking (Parker et  al. 2001) (Fig. 1.2c). Decreased levels of calcium-crosslinked pectin appear to reduce cell-­ cell adhesion, promoting the enzymatic isolation of single wall-less plant cells called protoplasts (Lionetti et al. 2015). Unlike at corner junctions, methylesterified pectins accumulate at regions between adjacent cells (Zamil and Geitmann 2017) (Fig. 1.2c). PMEs and PGs are required to degrade pectins and facilitate cell separation: for example, inhibiting PME activity prevents the separation of border cells from root tips (Wen et al. 1999). Downregulating PG activity also results in extended cell-cell adhesion in fruits (Posé et al. 2013). It is noteworthy that microspore separation requires the degradation of the primary cell walls of pollen mother cells, which envelop developing microspores. PMEs and PGs are required in this process, and mutations in PME and PG genes result in aberrantly connected tetrad pollen (Francis et  al. 2006; Rhee and Somerville 1998). Cell adhesion defects are also reported in HG synthesis mutants: perturbing HG chain elongation and methylesterification causes cell adhesion defects in Arabidopsis hypocotyls (Bouton et al. 2002; Krupková et al. 2007; Mouille et al. 2007). However, in some of these mutants,

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Fig. 1.3  Pectin functions in plant development and stress responses. Here, Arabidopsis is used as a model to show pectin functions in multiple developmental stages and stress responses. Pectins modulate organ elongation and expansion, like the elongation of hypoctyls, roots, and stems, and leaf expansion. Pectins function in organ morphogenesis by influencing organ patterning at apical meristems. During reproductive development, pectins mediate plant fertility by controlling pollen development, male-female interactions, fertilisation, and seed germination. The degradation of pectins also facilitates anther and silique dehicience, and organ abscission. For plant-environment interactions, pectins might triger cell wall intergity (CWI) signaling pathways via their interactions with FERONIA (FER) and other receptor-like kinases. Pectin degradation products, oligogalacturonides (OGs), might bind to Wall-associated kinase (WAK) receptors and trigger downstream immune responses (inset panel). PG polygalacturonase, PL pectate lyase, PR gene pathogen-­ related gene, ROS reactive oxygen species, PM plasma membrane. Figure made with BioRender

the cell adhesion defect does not directly result from the reduced HG content. Instead, lower HG levels appear to trigger pectin-related signalling pathways and abrogate cell adhesion (Verger et al. 2016). Asymmetric wall mechanics based on pectin asymmetries and cellulose orientation underlie cell anisotropic growth in rapidly growing tissues like dark-grown hypocotyls. In the epidermis of the dark-grown hypocotyl, methylesterified pectins localise in fast-growing axial walls, whereas demethylesterified pectins with calcium crosslinking are present in slow-growing transverse walls. Correlated with this asymmetric pectin distribution, axial walls show higher elasticity than transverse walls (Daher et al. 2018). Pollen tubes are useful models for studying tip growth in plant cells. In the pollen tube wall, methylesterified pectins are enriched at the apex, whereas demethylesterified pectins are only present along longitudinal walls (Fig. 1.2d). This gradient of pectin distribution is coupled with the spatial profile of wall extensibility, controlling pollen tube elongation (Fayant et al. 2010). To maintain this asymmetric distribution, PMEIs are restricted to the pollen tube apex and inhibit PME activity in this region (Chebli et al. 2012; Röckel et al. 2008). Impaired pollen tube growth is reported in mutant lines lacking functional PME, PG, and PAE genes (Gou et al. 2012; Huang et al. 2009a; Jiang et al. 2005). Additionally, RG-II

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is enriched at the pollen tube apex, and its borate cross-linking maintains boron concentrations, which are important for pollen tube elongation (Iwai et al. 2006). RG-I is widely present in the pollen tube wall, and its side chain structure prevents the tight apposition of HG polymers (Lehner et al. 2010). The development of complex cell shapes in plants can also be controlled by pectin level and status. Defective RG-I synthesis leads to helical growth of petal cells, distorted root hair cells, and twisted growth of petals and roots, which is independent from microtubule orientation (Diet et  al. 2006; Saffer et  al. 2017). In Arabidopsis, pavement cells develop from round cells into jigsaw puzzle piece-­ shaped cells with distinct neck (convex) and lobe (concave) regions. Lobe formation is closely related to the distribution of demethylesterified pectins (Fig. 1.2e). In the anticlinal wall, HG has been detected in nanofilament structures oriented perpendicular to the periclinal wall. Local HG demethylesterification is hypothesised to lead to radial swelling and asymmetric wall expansion, resulting in lobe formation (Haas et  al. 2020). In the periclinal wall, the accumulation of demethylesterified pectins at neck regions leads to higher wall stiffness than in lobe regions. This uneven mechanics restricts periclinal wall expansion in neck regions, and influences the orientations of microtubules and newly deposited cellulose as well (Altartouri et al. 2019). In addition, pectins facilitate dynamic cell processes, including stomatal opening and closing and mucilage secretion during seed imbibition. Stomatal dynamics are important for regulating gas exchange between plants and the environment. Wall flexibility in the guard cells that flank stomatal pores is mediated by pectic arabinan and HG (Amsbury et  al. 2016; Jones et  al. 2003; Rui et  al. 2017): for example, highly methylesterified pectin levels cause reduced dynamic range in stomatal movements (Amsbury et al. 2016), and mutation of a PG gene leads to inhibited stomatal closing, whereas excess HG degradation catalysed by overexpression of the same PG accelerates stomatal opening (Rui et al. 2017). During seed imbibition, mucilage is released from the locally ruptured outer primary wall and forms a gel-­ like structure at the seed surface. Pectin demethylesterification guides the localisation of peroxidases in the outer cell wall, facilitating wall loosening in that region (Francoz et al. 2019). Mucilage is abundant in unbranched RG-I, which is essential for its hydration properties. Insufficient side chain trimming on RG-I results in incomplete and slow mucilage release, as well as failed mucilage expansion (Arsovski et al. 2009; Dean et al. 2007). The multiple functions of pectins at the cellular level underlie their crucial roles in plant development (Fig. 1.3). Pectins function in anisotropic cell growth, contributing to organ elongation in hypocotyls, roots, and stems. Axial organ growth also shows a gradient in growth rate with the highest growth rate at the apical zone. In correlation with this growth gradient, pectin content, structure, and degree of methylesterification and acetylation also show apical-basal patterning in hypocotyls and inflorescence stems (Goldberg and Prat 1982; Phyo et al. 2017a). In inflorescence stems, the cell walls of the fastest growing segment contain more pectin and side chain structures, higher degrees of methylesterification and mobility, and less interaction with cellulose than in basal segments (Phyo et al. 2017a). In seedlings over-

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expressing a PG, the cell walls of hypocotyls show more methylesterified pectins and dispersed RG-I side chains, less calcium cross-linked HGs, and reduced interactions between pectin and cellulose (Phyo et  al. 2017b). Pectins also facilitate organ expansion. Altered RG-II structure with reduced borate cross-linking results in dwarf rosettes (O’Neill et al. 2001). Altered expression of PGs affects rosette leaf expansion (Xiao et al. 2014, 2017; Rui et al. 2017). Pectins modulate organ morphogenesis by mediating asymmetries in wall mechanics (Fig. 1.3). Leaf adaxial-abaxial polarity is essential for asymmetric leaf growth. In developing leaves, differential pectin methylesterification status among adaxial, middle, and abaxial domains is correlated with asymmetric wall mechanics, and further regulates leaf polarity. Ectopic expression of PME or PMEI genes in different leaf domains disturbs the asymmetry of wall mechanics, resulting in radially symmetrical leaves (Qi et  al. 2017). In the inflorescence meristem, pectin demethylesterification status in subepidermal layers modulates floral primordium initiation and maintains phyllotaxis. Inhibiting PME activity results in pin-like meristems and cylindrical flower primordia, whereas overexpressing PME causes the ectopic formation of floral organs (Peaucelle et al. 2008, 2011). During reproductive development, pectins are essential for the development of male and female gametophytes (Fig. 1.3). During early pollen development, methylesterified pectins are located on the outer layer of the microspore plasma membrane, as well as in the tapetum, which provides materials for pollen wall synthesis. In mature pollen walls, methylesterified and demethylesterified pectins are present at the inner layer (endintine) and outer layer (exintine) of the pollen intine, respectively (Aouali et al. 2001; Geitmann et al. 1995). Mutants for PME, PG, and PL genes show abnormal pollen intine development, reduced pollen activity, and delayed pollen germination (Huang et al. 2009a, b; Jiang et al. 2014; Leroux et al. 2015). To deliver sperm cells to the ovary, demethylesterified pectins enhance pollen tube adhesion to transmitting tracts (Jiang et al. 2005; Mollet et al. 2000). Along with the FERONIA receptor-like kinase, demethylesterified pectins at the filiform apparatus help to prevent multiple pollen tubes from penetrating a single female gametophyte (Duan et al. 2020). Apart from pectin modification, RG-II synthesis is necessary for male and female organ structure. Reduced RG-II content in a glucuronyltransferase mutant results in reduced pollen germination, branched pollen tube structure, and defective transmitting tracts, which affect the interaction between pollen tube and the ovary (Iwai et al. 2006). Pectins and their modification are essential for fruit ripening, organ abscission, and organ dehiscence, which are often accompanied by pectin degradation and loss of cell-cell adhesion (Fig. 1.3). In the early stage of fruit ripening, pectins undergo demethylesterification upon dramatically increased PME activity, and galactan and arabinan side chains are trimmed from RG-I (Draye and Van Cutsem 2008; Orfila et al. 2001). In the following stages, PGs and PLs depolymerise pectin and lead to fruit softening (Jiménez-Bermúdez et al. 2002; Quesada et al. 2009). The process of fruit softening can be manipulated by changing the expression of pectin-degrading enzymes: downregulating the expression of PG and PL genes in strawberry helps to maintain fruit firmness and elongates the duration of postharvest softening without

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influencing fruit weight and shape (Jiménez-Bermúdez et al. 2002; Quesada et al. 2009). In fact, the first genetically engineered food, the Flavr Savr tomato, benefited from this idea. By knocking down the expression of a PG, these tomatoes show longer shelf life and increased resistance to rotting (Bruening and Lyons 2000). During organ abscission and dehiscence, pectin degradation occurs in the cell wall and middle lamella in abscission zones and dehiscence zone. Cell detachment in these regions helps to remove senescent organs from plants, and is also essential for releasing mature pollen garins and seeds from anthers and siliques, respectively (Ogawa et al. 2009; Yang et al. 2018). In plant-environment interactions, pectins are involved in plant immune response to abiotic and biotic stresses (Fig. 1.3). During pathogen or parasitic plant invasion, the host cell wall acts as the first barrier to invasion and is degraded by exogenous pectin-modifying enzymes. Oligogalacturonides (OGs) are short fragments of degraded pectin, which can be released by endogenous, pathogen, or parasite PGs and PLs (Ferrari et al. 2013). During invasion, host PGIPs and PMEIs are upregulated to inhibit exogenous PG and PME activities, respectively. PGIPs can also regulate PG activity and trigger OG accumulation. OGs function as damage-­ associated molecular patterns, and OG signals are perceived by downstream receptors that activate defence responses, including the production of reactive oxygen species (ROS) and phytoalexins, callose accumulation, and the induction of expression of pathogen related (PR) genes (Benedetti et  al. 2015; Ferrari et  al. 2013; Gallego-Giraldo et  al. 2020). Wall-associated kinase 1 (WAK1) is one well-­ characterised OG receptor, and can bind OGs in a calcium-dependent manner (Decreux and Messiaen 2005). As the most mobile wall components, pectins serve as cell-wall integrity sensors, and can be bound by other types of cell wall integrity receptors. One such receptor is FERONIA, which directly interacts with pectins via its extracellular domain and might trigger signalling pathways to maintain cell-wall integrity during abiotic and biotic stresses (Feng et al. 2018; Vaahtera et al. 2019). Pectins are complex and intriguing constituents of the cell wall with wide-ranging localisation and function across the plant kingdom. For more information on these fascinating biomolecules, see Anderson (2016, 2019), Atmodjo et al. (2013), Daher and Braybrook (2015), Palin and Geitmann (2012), Peaucelle et al. (2012), Sénéchal et al. (2014), and Wolf and Greiner (2012).

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Iwai H, Hokura A, Oishi M et al (2006) The gene responsible for borate cross-linking of pectin rhamnogalacturonan-II is required for plant reproductive tissue development and fertilization. Proc Natl Acad Sci U S A 103:16592–16597 Jarvis MC, Briggs SPH, Knox JP (2003) Intercellular adhesion and cell separation in plants. Plant Cell Environ 26:977–989 Jiang L, Yang S-L, Xie L-F et  al (2005) VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract. Plant Cell 17:584–596 Jiang J, Yao L, Yu Y et al (2014) PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris. J Integr Plant Biol 56:1095–1105 Jiménez-Bermúdez S, Redondo-Nevado J, Muñoz-Blanco J et al (2002) Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene. Plant Physiol 128:751–759 Jones L, Milne JL, Ashford D, McQueen-Mason SJ (2003) Cell wall arabinan is essential for guard cell function. Proc Natl Acad Sci U S A 100:11783–11788 Kim S-J, Held MA, Zemelis S et al (2015) CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana. Plant J 82:208–220 Kong Y, Zhou G, Yin Y et al (2011) Molecular analysis of a family of Arabidopsis genes related to galacturonosyltransferases. Plant Physiol 155:1791–1805 Krupková E, Immerzeel P, Pauly M, Schmülling T (2007) The tumorous shoot development2 gene of Arabidopsis encoding a putative methyltransferase is required for cell adhesion and co-­ ordinated plant development. Plant J 50:735–750 Lampugnani ER, Khan GA, Somssich M, Persson S (2018) Building a plant cell wall at a glance. J Cell Sci 131:207373 Lehner A, Dardelle F, Soret-Morvan O et al (2010) Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil. Plant Signal Behav 5:1282–1285 Leroux C, Bouton S, Kiefer-Meyer M-C et  al (2015) Pectin methylesterase48 is involved in Arabidopsis pollen grain germination. Plant Physiol 167:367–380 Lionetti V, Cervone F, De Lorenzo G (2015) A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis. Phytochemistry 112:188–194 Matsunaga T, Ishii T, Matsumoto S et al (2004) Occurrence of the primary cell wall polysaccharide rhamnogalacturonan ii in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants. Plant Physiol 134:339–351 Mohnen D (2008) Pectin structure and biosynthesis. Curr Opin Plant Biol 11:266–277 Mollet J-C, Park S-Y, Nothnagel EA, Lord EM (2000) A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix. Plant Cell 12:1737–1749 Moore PJ, Swords KMM, Lynch MA, Staehelin LA (1991) Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. J Cell Biol 112:589–602 Mouille G, Ralet M-C, Cavelier C et  al (2007) Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain. Plant J 50:605–614 Nunan KJ, Scheller HV (2003) Solubilization of an arabinan arabinosyltransferase activity from mung bean hypocotyls. Plant Physiol 132:331–342 O’Neill MA, Eberhard S, Albersheim P, Darvill AG (2001) Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294:846–849 Ogawa M, Kay P, Wilson S, Swain SM (2009) Arabidopsis dehiscence zone polygalacturonase1 (ADPG1), ADPG2, and QUARTET2 are polygalacturonases required for cell separation during reproductive development in Arabidopsis. Plant Cell 21:216–233 Orfila C, Seymour GB, Willats WGT et al (2001) Altered middle lamella homogalacturonan and disrupted deposition of (1→5)-α-l-arabinan in the pericarp of cnr, a ripening mutant of tomato. Plant Physiol 126:210–221 Palin R, Geitmann A (2012) The role of pectin in plant morphogenesis. Biosystems 109:397–402

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Chapter 2

Pectin Structure David Ropartz and Marie-Christine Ralet

2.1  Introduction Pectin is a natural constituent of all terrestrial plants that is particularly abundant—together with hemicelluloses, cellulose, and low amounts of structural proteins—in the primary cell walls of eudicotyledons and non-graminaceous monocotyledons (Carpita and Gibeaut 1993). Once extracted from citrus peel or apple pomace, commercial pectin is widely used as gelling, thickening, stabilising and emulsifying agent in various food products such as jams, acidic milk drinks, ice creams, or salad dressings, and it is well agreed that the fine structure of pectin deeply affects its functionality and applicability (Willats et  al. 2006). The existence of a “jelly” in tamarind extract was discovered more than two centuries ago by the French pharmacist and chemist Louis-Nicolas Vauquelin (1790). The word “pectin” from the Greek πηκτός (“pêktós”, which means “thick”) was first used in 1825 by Braconnot, who had resumed Vauquelin’s work (Braconnot 1825a, b). Smolenski in 1923 was the first scientist to describe pectin as a polymer of galacturonic acid (GalA) and Kertesz (1951) defined pectin as a hetero-polysaccharide containing mainly partly methylesterified GalA together with some neutral sugars. In the 80s, the work of de Vries and co-workers was instrumental in showing that neutral sugars were present as side-chains arranged in blocks in so called “hairy regions” while >90% of the GalA residues could be isolated as chains comprising solely GalA (de Vries et al. 1981, 1982, 1983). Nowadays, it is widely accepted that pectin is a heterogonous macromolecule composed of interlinked distinct domains, the relative amount and structure of which vary according to the botanical origin, the organs and cell types considered, the stages of cellular development, and the precise location within the cell wall (Voragen et al. 2009). On top of this biological diversity, pectin structure varies broadly depending on the extraction D. Ropartz · M.-C. Ralet (*) INRAE, Biopolymères Interactions Assemblages, Nantes, France e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_2

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method used. Different pectin ­populations that are more or less strongly anchored in the cell wall co-exist and a high number of extraction steps is needed to extract all pectin (Broxterman 2018). Two main families of pectin structural elements are usually considered: galacturonans and rhamnogalacturonan I (RG-I). Galacturonans are made of a backbone of α-(1,4)-linked d-galacturonic acid (GalA) residues. This galacturonan backbone may be unbranched (homogalacturonan) or decorated with more or less complex side-chains. The backbone of RG-I is made of the diglycosyl repeating unit [2-α-l-­ Rha-(1,4)-α-d-GalA-(1] (Lau et al. 1985). Rhamnose (Rha) residues are ramified at O-4 (mainly) and O-3 (scarcely) positions with single to polymeric neutral sugar side chains that include arabinose (Ara) and galactose (Gal) residues in various combinations (Lau et al. 1985; Colquhoun et al. 1990). Four main types of polymeric side-chains are usually envisioned, arabinans, galactans, type I arabinogalactans (AG-I) and type II arabinogalactans (AG-II) (Yapo 2011). As has been briefly described above, pectin is an extremely complex polysaccharide composed of as many as eighteen distinct monosaccharides connected to each other through twenty different linkages. In addition, several of these monosaccharides can be chemically modified by O-ether or O-ester groups. Because of polydispersity and polymolecularity, analyses on whole macromolecules are usually not sufficient to reveal all the structural details of pectin. For structural investigation, pectin is thereby commonly degraded into oligosaccharides by chemicals or enzymes. Enzymatic digestion is usually preferred since it offers additional information thanks to the specificity of cleaving. Degradation enzymes are, however, not available for all linkages. Degradation products are further fractionated usually by chromatographic means. The isolated structural elements thereby recovered are in the analytical range of a broad set of techniques (Schols and Voragen 2002) and very valuable structural information has been obtained by chemical analyses and NMR in the 90s. This information has however been restricted to major oligosaccharides that could be purified in quantities large enough to allow full structural characterisation. In the past fifteen years, significant developments have led to a re-emergence of the use of mass spectrometry (MS) for the structural characterisation of oligosaccharides (Dong et  al. 2018). This review includes improvements that have been achieved in separation techniques (ultra-performance liquid chromatography (UPLC), capillary electrophoresis, ion mobility) and developments in activation approaches using electron, cation or photon interactions that are much more efficient than classical fragmentation methods (i.e., low energy collision induced (or activated) dissociation (LE-CI(A)D) for the determination of the fine structure of oligosaccharides. Very recent works have been conducted on instrument development for the structural characterisation of oligosaccharides based on ion activation (Ropartz et al. 2014, 2016, 2017), on the coupling of MS and infrared ion spectroscopy (Schindler et al. 2017; Mucha et al. 2018) and on high resolution ion mobility (Ujma et al. 2019; Ropartz et al. 2019). These approaches allow to shed new light on complex structures and mixtures of oligosaccharides, as can be found in pectin.

2  Pectin Structure

19

2.2  Galacturonans 2.2.1  Homogalacturonans Homogalacturonan (HG) also known as the “smooth region” of pectin, is a linear homopolymer of α-(1,4)-linked d-galacturonic acid (GalA) residues that can be methyl and acetyl-esterified (Fig. 2.1). It usually accounts for approximately 60% of the total pectin amount (Mohnen 2008; Atmodjo et al. 2013) but there are some exceptions—sugar beet or soybean pectin for instance—in which the relative amount of HG is much lower (Voragen et al. 1995). HG have been specifically isolated from several plant sources by exploiting the susceptibility to acid hydrolysis of the different glycosidic linkages (Thibault et al. 1993; Yapo et al. 2007; Ralet and Thibault 2009; Round et al. 2010) or by enzymatic means (Bonnin et al. 2002; Hellin et al. 2005). Depending on the method used for (1) pectin extraction, (2) HG isolation and (3) molar mass determination, the average degree of polymerisation (DP) ranges between 72 and 300. Ralet and Thibault (2009) isolated HG from pectin samples sequentially extracted from different plant sources: pineapple flesh (Ananas comosus, Bromeliaceae), leek (Allium porrum, Alliaceae), cucumber (Cucumis sativus, Cucurbitaceae), sugar beet root (Beta vulgaris, Amaranthaceae), fennel bulb (Foeniculum vulgare, Apiaceae), and lemon albedo (Citrus medica, Rutaceae) and showed that pectin samples encompassed various amounts of HG domains of very similar DP (70–100) and of low polydispersity, confirming the hypothesis of a HG length periodicity (Thibault et al. 1993). 2.2.1.1  Methyl-esterification In HG, GalA units are usually partially methyl-esterified at C-6 (Fig. 2.1) and not only the degree of methyl esterification (i.e., the number of moles of methanol per 100 moles of GalA), but also the distribution of non-esterified GalA residues on HG segments are key features for pectin functional properties (Willats et  al. 2006). Numerous investigations have thereby been devoted to understand the peculiar methyl-ester distribution patterns and their functional implications. The development of methods allowing characterising and quantifying degradation products

Fig. 2.1  Structure of homogalacturonans according to the symbol nomenclature for glycans (SNFG) introduced by Varki et al. (2015). Homogalacturonan is the dominant feature of pectin. It consists of partly methyl-esterified and, in some plant species, partly acetyl-esterified α-(1,4)linked d-galacturonopyranosyl chains with a degree of polymerisation 100–300. The amount and distribution of methyl- and acetyl-esters onto homogalacturonans is a key factor for pectin functionality

20

D. Ropartz and M.-C. Ralet

obtained by treating well-defined pectin samples with HG-degrading enzymes was instrumental in providing such information. The concept of degree of blockiness calculated from the amount of oligogalacturonates released quantified by high-­ performance anion-exchange chromatography or by capillary electrophoresis, has been developed. This concept allowed differentiating pectin encompassing HG domains exhibiting subtle differences in methylesterification patterns (Guillotin et al. 2005; Daas et al. 2000; Limberg et al. 2000; Ström et al. 2007; Ngouemazong et al. 2011; Ralet et al. 2012). Parallel electrospray ionisation multistage mass spectrometry (ESI-MSn) may be also used to provide information about the location of methyl-esterified GalA residues in oligogalacturonates of DP 3–10 (Körner et al. 1999; van Alebeek et  al. 2000; Quéméner et  al. 2003; Ralet et  al. 2009, 2012). Recent advances in mass spectrometry and separation techniques allowed exploring complex mixtures and characterising oligosaccharides of higher DP (identification up to DP14, structural characterization up to DP9) (Ropartz et  al. 2014). Model HGs were degraded by a pectin lyase and digestion products were characterised (Ropartz et al. 2014). The structure of each isomer was stated based on its methylation pattern. These approaches can bring different types of information: (1) the specificity of the enzyme and the tolerance of each subsite to the occurrence of methyl-esters can be stated, and (2) the structure of large oligosaccharides can be revealed, giving insights into parts of the polymers that are recalcitrant to enzymatic digestion. 2.2.1.2  Acetyl-Esterification In few plant species, GalA residues in HG domains are partially acetyl-esterified at O-2 and/or O-3 (Rombouts and Thibault 1986; Ishii 1997; Needs et  al. 1998; Perrone et  al. 2002) (Fig.  2.1) and this has a strong negative impact on gelation (Pippen et al. 1950; Kohn and Furda 1968; Kohn and Malovikova 1978; Renard and Jarvis 1999; Oosterveld et al. 2000a; Ralet et al. 2003). The distribution of acetyl groups onto HG segments has been particularly studied in sugar beet. Keenan et al. (1985) showed by nuclear magnetic resonance (NMR) that acetyl groups could be attached on any of the available ring positions (O-2 and O-3) of GalA residues. Ralet et al. (2005, 2008) and Remoroza et al. (2014) used combinations of pectin-­ degrading enzymes to generate partly methyl esterified and acetyl esterified oligogalacturonates that were further separated by chromatographic means and analysed by ESI-MSn. The large variety of oligogalacturonates that were identified and quantified revealed that (1) the occurrence of O-2 and O-3 acetyl esterification in roughly similar amounts, (2) the absence of 2,3-di-O-acetylation, and (3) the scarcity of GalA residues that are both methyl- and acetyl-esterified (Ralet et al. 2005). Pectins with high degree of methyl esterification (HM) having different patterns of ester distribution could be also discriminated (Remoroza et al. 2014). A blockwise distribution of acetyl groups was evidenced (Ralet et  al. 2008) and, in commercially-­ extracted sugar beet pectin, blocks of (1) non-esterified, (2) partly methyl- and acetyl-esterified, and (3) highly methyl- and acetyl-esterified GalA residues were

2  Pectin Structure

21

identified (Remoroza et al. 2014). The balance between intra-chain heterogeneity (i.e., the different blocks that are present within a single macromolecule) and inter-­ chain heterogeneity (some pectin molecules are very highly acetyl-esterified and others are not) is however very difficult to appraise.

2.2.2  G  alacturonans Substituted with More or Less Complex Side-Chains HG can be more or less heavily substituted at O-2 and/or O-3 by monomers or dimers of apiose or xylose leading to apiogalacturonan (AGA, Fig. 2.2a) or xylogalacturonan (XGA, Fig. 2.2b), respectively. It can also be substituted with complex side-chains to form rhamnogalacturonan II (RG-II, Fig. 2.2c).

Fig. 2.2  Structure of the side-chains of homogalacturonans (SNFG). (a) apiogalacturonan, (b) xylogalacturonan, (c) rhamnogalacturonan-II. More or less complex side-chains can be attached to homogalacturonan. Rhamnogalacturonan-II is a key substituted galacturonan made of a short backbone branched by up to 5 side-chains varying in complexity from single arabinose unit to highly heterogeneous nonasaccharides

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2.2.2.1  Apiogalacturonan Apiogalacturonan (AGA, Fig. 2.2a) is restricted to some aquatic plants, the duckweeds (Lemnoideae) and the marine seagrasses (Zosteraceae) (Hart and Kindel 1970; Ovodov et al. 1971; Longland et al. 1989; Gloaguen et al. 2010; Avci et al. 2018). In AGA, β-d-apiofuranosyl residues are linked to O-2 and/or O-3 of GalA residues as monomers or as the dimer [β-d-Apif-(1,3′)-β-d-Apif-(1)]. The degree of substitution of HG by apiose varies from 25 to 80% (Hart and Kindel 1970; Ovodov et al. 1971). AGA can represent up to 20% of non-cellulosic cell wall polysaccharides in the green fronds of giant duckweeds (Longland et al. 1989). 2.2.2.2  Xylogalacturonan Xylogalacturonan (XGA, Fig.  2.2b), an HG substituted solely at O-3 by xylose monomers or dimers, has been identified in several plant samples among which duckweeds (Hart and Kindel 1970), apple and watermelon fruits (Schols et  al. 1995a; Zandleven et  al. 2006; Mort et  al. 2008), potato tuber (Zandleven et  al. 2006), pea hulls (Le Goff et al. 2001) and Arabidopsis thaliana leaves and stalks (Zandleven et al. 2007). Dimeric side chains of xylose have been shown to contain 1,4-linked xylose residues (Zandleven et  al. 2006) but 1,2-linked and 1,3-linked xylose residues have also been identified (Le Goff et  al. 2001; Nakamura et  al. 2002). Methylesterification of XGA has been reported in apple (Schols et al. 1995a). Both the ratio of Xyl monomers and dimers and the degree of substitution of GalA by Xyl vary according to plant samples. A recent study highlighted that the diversification of the Lemnoideae was accompanied by a reduction in the abundance of cell wall AGA and an increase in XGA (Avci et al. 2018). 2.2.2.3  Rhamnogalacturonan II Rhamnogalacturonan II (RG-II, Fig.  2.2c) is a far more complex substituted-HG than AGA or XGA since it encompasses thirteen different sugars and twenty one distinct glycosidic linkages arranged as a backbone formed by nine partially methyl-­ esterified GalA residues (from none to three) (Ishii and Kaneko 1998) substituted by different side chains termed A–F (Ndeh et al. 2017). Large Side-Chains Side-chains A and B encompass six to nine sugars and are both bound to the HG backbone through a (2,1) β-d-apiofuranose. Although the structure of RG-II is considered as highly conserved throughout the plant kingdom, some heterogeneity may occur within these side-chains. Side-chain A was first described by Stevenson et al. (1988) in Ficus sycomorus as an oligosaccharide composed of eight different monosaccharides (Fig. 2.2c). The heterogeneity in the structure of side-chain A comes firstly from the α-l-Galp residue that can be replaced by an α-l-Fucp, not only in

23

2  Pectin Structure

the Arabidopsis thaliana mutant mur1 (Reuhs et al. 2004), but also in the wild-type plants (Pabst et al. 2013). Additionally, side-chain A contains three uronic acids, one α-d-GalAp, one β-d-GalAp and one β-d-GlcAp, that can be methyl-esterified and/ or methyl-etherified (Pabst et al. 2013; Buffetto et al. 2014). Side-chain A is involved in RG-II dimerisation through a boron covalent linkage via a di-ester bond between two β-d-apiofuranose units (O-2 and O-3). The three uronic acids residues are able to chelate cations and methyl-esterification that can occur on the β-d-GlcAp unit may modulate this chelation property. Side-chain B is particularly heterogeneous (Table 2.1). Table 2.1  Structural variability of side-chain B in rhamnogalacturonan-II

Reference Spellman et al. (1983) Thomas et al. (1989) Whitcombe et al. (1995) Kaneko et al. (1997) Shin et al. (1998) Ishii and Kaneko (1998) Vidal et al. (2000) Glushka et al. (2003) Matsunaga et al. (2004) Reuhs et al. (2004) Seveno et al. (2009) Pabst et al. (2013) Buffetto et al. (2014) Sun et al. (2019)

Plant origin Acer pseudoplatunus

Glycosyl substitutions (2-1) (2-1) α-l-­ β-l-­ Rhap Araf X –

Chemical modifications Acetyl Methylether (2-1) (3-1) (3-1) α-l-­ 3-α-l-­ 2-O-Me-­ α-l-­ α-l-­ Rhap Acef α-d-Fucp Rhap Rhap – – – – –

Oryza sativa

X

–

–

–

–

–

–

Acer pseudoplatunus

X

X/–

–

X/–

X/–

–

–

Phyllostachys edulis

X

–

–

–

–

–

–

Panax ginseng

X

X/–

X/–

X/–

X/–

–

–

Beta vulgaris

–

–

–

X/–

X/–

–

–

Red wine

X

X/–

X/–

X/–

–

–

Arabidopsis thaliana

X

X

X

X/–

X/–

–

–

X/–

X/–

X/–

X/–

X/–

X/–

–

–

–

–

–

–

Lycophyte X PteridophyteBryophytes Arabidopsis thaliana X

a

Arabidopsis thaliana

X/–

–

–

–

–

–

–

Arabidopsis thaliana

X

X/–

X/–

–

–

–

–

Vitis vinifera Merlot

X

X/–

X/–

X/–

X/–

–

–

Panax ginseng

X

X/–

X/–

X/–

X/–

–

–

X detected, – not detected a Additional rhamnose was detected but not localised

24

D. Ropartz and M.-C. Ralet

It was first described in Ficus sycomorus as a heptasaccharide, the main chain being made of a β-d-Apif-(3,1)-β-l-Rhap-(3,1)-α-l-Aceric acid-(2,1)-β-d-Galp-(2,1)-2-OMe-α-l-Fucp. The Galp residue was substituted by a (4,1)-α-L-Arap unit itself substituted by a (2,1)-α-l-Rhap residue (Spellman et al. 1983). Depending on the botanical origin, the stage of development and the organ considered, the number of constitutive sugar residues may vary. Hexa- to nonasaccharides were observed based on the substitution of the laterally branched α-L-Arap residue (Fig. 2.2). Whitcombe et al. have described an octasaccharide with a (2,1)-β-l-Araf linked to the laterally branched additional α-l-Rhap unit. These authors also described for the first time the occurrence of two acetylation sites on the α-l-Aceric acid and on the 2-O-Me α-d-Fucp. Finally, in 1998, Shin et al. described in Panax ginseng a nonasaccharide resulting from a second α-l-Rhap unit branched at C-3 of the β-l-Arap residue. It is difficult to conclude about the existence of the low DP species in vivo, as α-l-Rhap residues being very labile according to the extraction conditions and the analytical methods used. As for sidechain A, 2-O-Me-α-d-Fucp can be replaced by 2-O-Me-l-Galp in the Arabidopsis thaliana mur1 mutant (Reuhs et  al. 2004). In lycophytes, pteridophytes and bryophytes, α-l-Rhap units can be methyl-etherified at O-3 (Matsunaga et al. 2004). Short Side-Chains Side chain E is composed of a unique residue of α-l-Araf linked to a d-GalAp at O-3 (Buffetto et al. 2014). This modification is termed side chain F when it occurs on a d-GalA unit that is also substituted by side-chain A. Side-chains C and D are dimers that are branched at O-3 of a d-GalA units. They are composed of a α-3deoxy-d-manno-­ 2-octulosonic acid (4,1) α-l-Rhap and an α-3-deoxy-d-lyxo-2heptulosonic acid (5,1) β-l-Araf, respectively. In the fractionation scheme used by Buffetto et al. (2014), these side chains are co-eluted with longer ones (co-elution of A and D and of B and C) (Ropartz 2015).

2.3  Rhamnogalacturonan I RG-I was pictured as a long sequence of alternating l-Rha and d-GalA residues (Fig.  2.3a), Rha residues being substituted with a variety of l-arabinosyl- and d-galactosyl-containing side-chains (O’Neill et  al. 1990, Fig.  2.3b–d). Fucose, glucuronic acid and 4-O-methyl glucuronic acid residues may also be present in small amounts. RG-I usually accounts for 20–35% of the total pectin amount (Mohnen 2008) but can, for certain plant sources such as soybean, make up to 75% of pectic polysaccharides (Voragen et al. 2009).

2.3.1  Rhamnogalacturonan I Backbone The backbone of RG-I is made of the alternating diglycosyl repeating unit [2-α-l-­ Rhap-(1,4)-α-d-GalAp-(1] (McNeil et al. 1980, 1984; Lau et al. 1985, Fig. 2.3a).

2  Pectin Structure

25

Fig. 2.3  Structure of Rhamnogalacturonan I (SNFG). (a) Backbone, (b) Arabinans, (c) Type I arabinogalactans, (d) Type II arabinogalactans. Rhamnogalacturonan I is the second major element of pectin. Its backbone is made of strictly alternating galacturonic acid and rhamnose residues, the latter being branched by arabinose- and galactose-containing side-chains of diverse complexity

Whole RG-I samples isolated from various plant sources usually exhibit Rha/ GalA ratios < 1 (Schols and Voragen 1994; Cornuault et al. 2015; Buffetto et al. 2015). Values very close to 1 have however been reported (Konno et  al. 1986; Yapo et al. 2007; Ralet and Thibault 2009) and the series of homologous oligomers recovered after controlled acid hydrolysis of pectin samples from apple, sugar beet and citrus all presented strictly alternating sequences (Renard et  al. 1995). The presence of RG-I domains in which single Rha residues alternate with two or three consecutive GalA residues is therefore rather implausible. Unlike HG domains, RG-I backbone seems to exhibit a DP that varies depending on the plant source. An average DP of up to 300 repeats has been estimated for RG-I isolated from suspension-cultured sycamore cell walls (McNeil et al. 1984). Ralet and Thibault (2009) reported values of 60 Rha-GalA repeats for RG-I isolated from citrus albedo and 120 for RG-I isolated from sugar-beet. Acetylation is much more frequent in RG-I than in HG, and RG-I domains isolated from numerous plant species appeared highly acetylated at O-2 or O-3 of the GalA units (Komalavilas and Mort 1989; Lerouge et  al. 1993; Schols and Voragen 1994; Normand et al. 2010; Remoroza et al. 2012). Ishii showed that some GalA residues were 2,3-di-O-acetylated in RG-I oligosaccharides isolated from potato tuber and bamboo shoot cell walls (Ishii 1997). In okra, unusual acetylation at O-3 of Rha residues has been also shown (Sengkhamparn et al. 2009). Finally, to our knowledge, no evidence for methylesterification of the RG-I domain has ever been reported.

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2.3.2  Rhamnogalacturonan I Side-Chains RG-I is also known as the pectin “hairy region” since GalA but mostly Rha residues are substituted by different side-chains varying in their length and composition. GalA residues typically are not substituted and only one study showed that approximately 2% of the GalA residues in the RG-I backbone were substituted at O-3 by single β-d-glucuronic acid residues (Renard et  al. 1999). In contrast, 20–80% of the Rha residues are substituted at O-4 with side-chains in which Ara and Gal predominate (Ridley et  al. 2001). The proportion of side-chains, their composition, length and degree of branching vary enormously not only depending on plant sources, organs and tissues (Lerouge et al. 1993) but also on developmental stage (Willats et  al. 2001) and on the isolation method used (Schols et  al. 1995b). For instance, arabinan-rich pectins are particularly abundant in apple, sugar-beet and carrot while type I arabinogalactan-rich pectins are commonly found in citrus, lupin and potato and type II arabinogalactan-rich pectins have been found in ginseng (Sun et al. 2019). Further, galactan and arabinan epitopes can occur in different zones of organs and even at distinct locations within a single developing organ (Willats et al. 2001). Finally, for a given cell wall material, pectins that were more difficult to extract were more substituted with side-chains than the more easily extractable fractions (Schols et al. 1995b). Side-chains consisting in single Gal units have been reported for RG-I oligomers that have been isolated without using any side-chain degrading enzymes (Schols et al. 1995b; Oosterveld et al. 2000b; Sun et al. 2019). In okra, specifically, α-Gal substitutions were identified (Sengkhamparn et al. 2009). Polymeric side-chains commonly consist in arabinans and type I and II (arabino)-galactans. 2.3.2.1  Arabinans Arabinans are made of a backbone of α-(1,5)-linked l-Araf residues that are substituted at O-2 and/or O-3 with single α-l-Araf units or with short chains of α-(1,3)linked l-Araf units (Yapo 2011, Fig.  2.3b). Sugar-beet arabinans have been particularly studied over the years. They consist of a long backbone of up to sixty or seventy Ara residues (Oosterveld et al. 2002) one third of which being branched at O-3, mainly with single Araf units (Guillon and Thibault 1989). The presence of minor amounts of double branching at O-2 and O-3 has been further evidenced (Westphal et al. 2010). Occasionally, arabinans substituted with single Galp units or with up to four or five β-(1,4)-linked Galp residues have been isolated in soybean, sugar-beet or potato (Sakamoto and Sakai 1995; Nakamura et al. 2002; Øbro et al. 2004). Arabinans branched to a much lower extent than in sugar-beet have been also observed in ginseng root and in potato tuber (Sun et al. 2019; Øbro et al. 2004).

2  Pectin Structure

27

2.3.2.2  Type I (Arabino)-Galactans Type I (arabino)-galactans encompass a backbone of β-(1,4)-linked d-Galp residues that may be lowly substituted at O-3 by β-d-Galp or α-l-Araf single units or by short α-(1,5)-linked l-Araf chains (Aspinall et al. 1967; Morita 1965a, b, Fig. 2.3c). Variants of this basic structure have been reported such as (1) termination of the main chain at O-6 with a Galp residue (Lau et al. 1987), (2) termination of the main chain with an Arap residue at O-4 or a Fuc residue at O-2 (Huisman et al. 2001; O’Neill et al. 1990), (3) insertion of a single Araf or a short α-(1,5)-linked l-Araf chain within the β-(1,4)-linked d-Galp main chain (Huisman et al. 2001; Buffetto et al. 2015), and (4) β-(1,3) galactosyl interruption of the main chain (Hinz et al. 2005). An average chain length in the range of 45–50 was estimated in soybean (Huisman et al. 2001). 2.3.2.3  Type II Arabinogalactans Type II arabinogalactans are highly branched side-chains characterised by a backbone of β-(1,3)-linked d-Galp residues substituted at O-6 with single Galp residues or with short chains of β-(1,6)-linked d-Galp residues, which can in turn be substituted at O-3, O-4 and/or O-6, by Ara-containing chains (Yapo 2011, Fig.  2.3d). Type II arabinogalactans have been recently identified in ginseng (Sun et al. 2019) and it has been shown that the (1,3)-linked galactan backbone was substituted, mainly at O-6 but also at O-4, (1) with short (1,6)-linked galactan chains terminated by single Araf units, and (2) directly with single Araf residues. 2.3.2.4  Ferulic Acid In species from the Amaranthaceae family such as sugar-beet and spinach RG-I side-chains are esterified by ferulic acid residues (Ishii and Tobita 1993; Colquhoun et al. 1994; Levigne et al. 2004; Quéméner and Ralet 2004). Ferulic acid moieties are mainly ester-linked to O-2 of Ara residues from the α-(1,5)linked l-Araf arabinan backbone and to O-6 of Gal residues from the β-(1,4)linked d-Galp type I galactan backbone but a more peripheral location at O-5 of Ara residues from the α-(1,5)-linked l-Araf arabinan backbone has been also identified. Ferulic acid residues can undergo in vivo oxidative coupling reactions to form dehydrodimers, thereby covalently cross-linking the polysaccharides they esterify (Fry 1986). Such cross-links have been identified in sugar-beet pectin (Ralet et al. 2005).

28

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2.3.3  Side-Chain Intra- and Inter-Molecular Distribution Treatment of “hairy regions” with rhamnogalacturonan hydrolase allowed to evidence that the different types of side-chains were not randomly distributed over the RG-I backbone. Indeed, incubating de-esterified apple or ginseng pectin with rhamnogalacturonan hydrolase released RG-I oligosaccharides based on a GalA2Rha2 and a GalA3Rha3 backbone (Schols and Voragen 1994; Sun et al. 2019). Most of the RG-I oligomers recovered contained no side-chains and single unit Gal side-chains even though single units or short Ara chains were also detected in ginseng (Sun et al. 2019). Since Rha-GalA oligosaccharides substituted solely by Ara residues were identified (O’Neill et  al. 1990; Sun et  al. 2019), a direct linkage between Ara and Rha is plausible although a single Gal unit or short type I galactan anchor seem to be usually necessary for Ara and arabinan branching onto RG-I backbone (Buffetto et al. 2015). Next to these lowly branched rhamnogalacturonan hydrolase-­degradable regions, highly branched arabinan- and type II arabinogalactan-rich regions that are not rhamnogalacturonan hydrolase-degradable have been identified (Schols and Voragen 1994; Sun et al. 2019). It has been hypothesised that these side-chains are flexible and long enough to wrap around the RG-I backbone, hindering enzymatic breakdown of the RG-I backbone by rhamnogalacturonan hydrolase (Willats et al. 2001; Voragen et al. 2009). The intra- and inter-molecular distribution of the different domains (lowly and highly branched) is extremely difficult to characterise and virtually nothing is presently known about the specific location of individual side-chains along the backbone. The co-existence of different “hairstyles” within a single pectin molecule remains speculative (Voragen et al. 2009).

2.4  Connection Between Pectin Domains It is now fully admitted that pectin is a highly complex macromolecule in which several domains are covalently linked to each other. It is however particularly challenging to accommodate all available structural information into a universal model structure. Currently, two models are under debate (1) the smooth and hairy regions model established by de Vries et  al. (1981) and updated by Schols and Voragen (1996) (Fig.  2.4) and (2) the RG-I backbone model proposed by Vincken et  al. (2003) (Fig. 2.5). The smooth and hairy regions model proposes that HG domains alternate with “hairy” regions, the latter including three subunits: subunit I: XGA; subunit II: stubs of RG-I backbone substituted by arabinan side-chains (or AG-II, Sun et al. 2019); and subunit III: rather short RG-I stretches (four or six residues) that are unsubstituted or substituted by Gal single units (Schols and Voragen 1996). In contrast, the RG-I backbone model positions HG and XGA as side-chains of RG-I (Vincken et al. 2003). Coenen et al. (2007) are, according to our knowledge, the

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Fig. 2.4  Pectin smooth and hairy region model. The smooth region consists of homogalacturonan. The hairy region consists of three types of subunits: subunit I made of xylogalacturonan, subunit II made of arabinan- and type II arabinogalactan-rich RG-I and subunit III made of lowly substituted RG-I short stretches. Adapted from Schols and Voragen (1996)

only group succeeding in isolating and characterising chimeric oligosaccharides with a HG/XGA segment linked to a RG-I backbone one. A GalA trimer and a Xylsubstituted GalA trimer covalently linked to a short RG-I moiety were identified and H1-NMR unambiguously showed that the GalA trimer was α-(1,2)-linked to the RG-I moiety. MSn experiments further showed that the RG-I moiety was always located at the reducing end of the chimeric oligosaccharides. These results are undoubtedly in favour of the “smooth and hairy regions” model even though this model does not fulfil all the experimental results obtained so far. In particular, it has been calculated that there are in average seventeen HG domains for one RG-I domain in lemon acid-­extracted pectin and eight HG domains for one RG-I domain in sugar beet acid-­extracted pectin taking into account (1) the partitioning of GalA between HG and RG-I domains, and (2) the length of these domains (Ralet and Thibault 2009). A similar “excess of GalA” was noticed by Yapo et  al. (2007) and by Coenen et al. (2007). It is clear that there is a huge surplus of HG domains to build up a pectin macromolecule following the “smooth and hairy regions” model; the “RG-I backbone” model shall therefore not be excluded irrevocably (Coenen et al. 2007).

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Fig. 2.5  Pectin RG-I backbone model. Homogalacturonan and xylogalacturonan (yellow) together with arabinans, and arabinogalactans (green) are side-chains of the RG-I backbone

2.5  Conclusions Pectin is a highly complex macromolecule exhibiting a high degree of intra- and inter-molecular heterogeneity. When extracting pectin from a given plant sample, different populations that were located in specific tissues or even in specific spots within a single cell wall are co-extracted and mixed. Studying pectin fine structure implies to be able to master extraction conditions and implement adequate chemical and enzymatic tools to generate oligomers that can be separated and fully analysed. These can further be used to generate probes such as monoclonal antibodies to locate specific motifs in muro. To date, complete deconstruction of pectin into analysable oligomers has not been achieved and non-degradable fractions whose structure is unclear always remain. These fractions, encompassing in particular GalA surplus, should be targeted for getting a better understanding of the architecture of pectin macromolecules.

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Chapter 3

Pectin Degrading Enzymes Estelle Bonnin and Jérôme Pelloux

3.1  Introduction Pectins are complex polysaccharides present in the primary cell wall, their main characteristic being their high content of partly methyl-esterified galacturonic acid (GalA), and the presence of neutral sugars on the side chains. The seventeen different monosaccharides and more than twenty different linkages make it a very complex macromolecule, whose enzymatic degradation requires many different enzymes related to the narrow specificity of each. Enzymes involved in pectin degradation catalyse different types of bonds. Hydrolases split a covalent bond by adding a molecule of water while the lyases catalyse β-elimination reactions without the addition of a water molecule. The former comprise particularly the glycosidases and the esterases, and belong to the group 3 of the Enzyme Commission (EC 3.). The latter belong to the group 4 (EC 4.) and produce a double bond on the newly formed non-­ reducing end. Polysaccharide-degrading enzymes can be distinguished as endo- or exo-enzymes. The endo-enzymes cut linkages randomly inside the chain and most often are less active when the degree of polymerisation decreases. In contrast, exo-­ enzymes have the highest affinity for the terminal bond on the reducing- or non-­ reducing end of the chain. Enzymes related to synthesis, degradation, or modification of oligo- and polysaccharides are classified in the CAZy (for Carbohydrate-Active enZymes) database (www.cazy.org). Glycoside-hydrolases (GH), polysaccharide-­ lyases (PL), carbohydrate-esterases (CE), glycoside-transferases and carbohydrate-­ binding modules are grouped into structurally- and catalytically-related families. E. Bonnin (*) INRAE, UR BIA Biopolymères Interactions Assemblages, Nantes, France e-mail: [email protected] J. Pelloux BIOPI Biologie des Plantes et Innovation SFR Condorcet, Université de Picardie, Amiens, France e-mail: [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_3

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This chapter focuses on highlighting the diversity of pectin-related enzymes by describing their structural diversity, the regulation of their expression, their substrate specificity and modes of action. Considering the diversity of pectin structure, enzymes will target a large diversity of linkages and/or polysaccharides, acting either on the backbone or on the side chains of pectin. These changes in pectin chemistry have dramatic consequences on polymer rheology, and ultimately the cell wall, affecting plant development, plant susceptibility to pathogens as well as the quality of end-products. Despite having being studied for a number of years, understanding the diversity of pectin-related enzymes is still a major challenge.

3.2  Homogalacturonan-Degrading Enzymes Homogalacturonan (HG) backbone is depolymerised by different enzymes according to the recognition site on the GalA chain (Table  3.1; Fig.  3.1a). The linkage between two non-esterified GalA is recognised by polygalacturonases (PG) and pectate lyases (PL), whereas the linkage harboring an esterified GalA at the right side is recognised by pectin lyases (PNL). PL and PNL are often referred as to PLL (Pectin Lyase-Like). Fig. 3.1  Site of action of pectin degrading enzymes. (a) Homogalacturonan degrading enzymes, (b) Rhamnogalacturonan degrading enzymes. Grey circle: galacturonic acid, grey square: rhamnose, purple triangle: galactose, brown diamond: arabinose, orange square: methyl group, and red diamond: acetyl group. PG polygalacturonase (endo/ exo), PL pectate lyase (endo/exo), PNL pectin lyase, PME pectin methylesterase, PAE pectin acetylesterase, endo-Gal endogalactanase, β-Gal β-galactosidase, endo-A endo-arabinanase, AF arabinofuranosidase, RgH RG-hydrolase, RgL RG-lyase, RgAE RG-acetylesterase

Substratea HG–LM Pectin HG–LM Pectin HG–LM Pectin RG-I–Pectin RG-I–Pectin RG-I–Pectin Xylogalacturonan 1,4 Galactan 1,3-6 Galactan 1,3-6 Galactan Oligogalactosides Arabinan Oligoarabinosides HG HG HM Pectin RG-I RG-I Pectin Pectin Pectin Feruloylated oligosides

CAZya family GH28 GH28 GH28 GH28 GH28 GH28 GH28 GH53 GH16 GH5-30 GH1-2-35-42 GH43 GH43-51-54-62 PL1-2-9 PL1-2-9 PL1 PL4 PL11-26 CE8 CE12 CE12 CE1

Pectic acid + methanol Pectic acid + acetic acid RG-I + acetic acid Oligosides + ferulic acid

Unsaturated OligoGalA Unsaturated OligoGalA Unsaturated MeOligoGalA Unsaturated OligoRG Unsaturated OligoGalA

OligoGalA GalA GalA2 OligoRG Rhamnose GalA Xylosylated OligoGalA 1,4 Oligogalactosides 1,3-6 Oligogalactosides 1,3-6 Oligogalactosides Galactose Oligoarabinosides Arabinose

Product

a

GH glycosyl hydrolase, PL polysaccharide lyase, CE carbohydrate esterase, HG homogalacturonan, RG-I rhamnogalacturonan I, LM low methoxy, HM high methoxy, GalA galacturonic acid

Table 3.1  Enzymes involved in pectin degradation Enzymes EC number Hydrolases Endopolygalacturonase 3.2.1.15 Exopolygalacturonase 3.2.1.67 Exo-poly-α-D-galacturonosidase 3.2.1.82 Rhamnogalacturonan hydrolase 3.2.1.171 Rhamnogalacturonan rhamnohydrolase 3.2.1.174 Rhamnogalacturonan galacturonohydrolase 3.2.1.173 Xylogalacturonanhydrolase 3.2.1.– Endo (1,4) galactanase 3.2.1.89 Endo (1,3) galactanase 3.2.1.181 Endo (1,6) galactanase 3.2.1.164 β-galactosidase 3.2.1.23 Endoarabinanase 3.2.1.99 α-L-arabinofuranosidase 3.2.1.55 Lyases Endopectate lyase 4.2.2.2 Exopectate lyase 4.2.2.9 Endopectin lyase 4.2.2.10 Rhamnogalacturonan endolyase 4.2.2.23 Rhamnogalacturonan exolyase 4.2.2.24 Esterases Pectin methyl esterase 3.1.1.11 Pectin acetyl esterase 3.1.1.– Rhamnogalacturonan acetyl esterase 3.1.1.– Feruloyl esterase 3.2.1.73

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Polygalacturonases (PG)

Endo-polygalacturonases (Endo-PG, EC 3. 2.1.15) and exo-polygalacturonases (Exo-PG, EC 3.2.1.67) generally catalyse the hydrolysis of the α-(1–4) glycosidic bond between two adjacent D-GalA units in non-methylated stretches of HG.  In plants, they are synthesised in the endoplasmic reticulum before being modified in the Golgi apparatus and secreted in the cell wall where they contribute to the changes in the pectin chemistry and mechanics during plant growth. In tomato fruit, they notably contribute to ripening and to the loss of firmness during storage (Van Dijk et  al. 2006). In contrast, apple pectins do not appear to be major targets for the ripening-­involved enzymes. Nevertheless, PG1 expression levels have been associated with softening in a range of cultivars (Wakasa et al. 2006) and suppression of PG1 gene expression in transgenic apple plants results in a firmer fruit (Atkinson et al. 2012). PG play a key role in the regulation of plant development such as root/ hypocotyl growth, stomata functioning, and pollen formation, as has recently been highlighted in a number of studies in Arabidopsis (Rhee et al. 2003; Ogawa et al. 2009; Xiao et al. 2014, 2017; Rui et al. 2017). Phytopathogenic organisms, including microorganisms and parasitic plants, also produce PG. They contribute to host colonisation by degrading the physical barrier of cell wall. In herbivorous insects, although it has long been assumed that pectin degradation is ensured by enzymes from the digestive microflora, advances in genome sequencing and bioinformatics analysis have recently revealed the presence of endogenous pectin-related enzymes (Kirsh et al. 2016). In the CAZy database, the GH family 28 includes all the PG. As reviewed by Yang et al. (2018), they all share the four amino-acids segments NTD, DD, GHG and RIK in their active site. The aspartic acid residues in the two first segments are the catalytic acids. From the reported structures of microbial PG, they display a β-helix 3D structure. The active site of a typical PG is a tunnel-like binding cleft with which the enzyme binds the polysaccharide to produce oligosaccharides with variable degrees of polymerisation (Shimizu et al. 2002). In contrast, in a typical exo-PG, inserted stretches of amino acids transform this open-ended channel in a closed pocket only able to attack the non-reducing end of the pectic oligomers (Abbott and Boraston 2007). In the genomes of the mammalian intestine bacteria, enzyme-coding sequences are grouped together in polysaccharide utilisation loci (PUL) (Flint et al. 2008). In the gut bacteria Bacteroides xylanisolvens, six PUL are devoted to pectin degradation, including one particularly involved in HG breakdown (Despres et al. 2016). Whatever the source considered, PG are most often the products of large multigenic families. For instance, even in monocotyledonous plants, such as Brachypodium dystachion where pectin content is low, more than forty sequences have been annotated as putative PG (The International Brachypodium Initiative 2010). All these isozymes exhibit different specificities, which are likely to play a key role in the adaptation to different environments. As an example, all PG isoforms in the model plant Arabidopsis thaliana have a predicted pI between 4.55 and 10.26 (data extracted from www.arabidopsis.org). This would regulate their activity according to the pH and ionic microenvironment of the cell wall. To a lesser extent, the fila-

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mentous fungus Aspergillus niger produces seven endo-PG with different optimal conditions for pH and temperature. In most cases, optimal pH for PG is in the acidic range, from 3 to 6. This optimum can be modulated by the presence of ions. Monovalent cations such as Na+ affect the optimal pH by screening the charges of the carboxylic groups. Divalent cations such as calcium interact with the HG chains to form dimers and thus modify the availability of the substrate to the enzyme. Even if the main feature of PG is to break down linkages between non-substituted GalA, the isoforms of A. niger vary in their tolerance towards methyl substituents on the GalA chain. Exo- and endo-PGs can also vary in their tolerance towards acetyl ­substituents (Sakamoto et al. 2002; Bonnin et al. 2003). Xylose also occurs as substituent in xylogalacturonan. Some exo-PG are able to release the dimer Xyl-GalA (Sakamoto et al. 2002) while endo-enzymes named xylogalacturonases work within the chain (Zandleven et al. 2005, 2006). When attacked by pathogens the host plant secretes in the cell wall polygalacturonase-­inhibiting proteins (PGIP) dedicated to the inhibition of polygalacturonases. From the current knowledge, PGIP are not involved in the regulation of plant PG activity but target the microbial PG with various specificities. PGIP belong to a large family of leucine-rich proteins and the residues involved in the β-strand/β-turn motif were shown to be crucial for their affinity and specificity to PGs. The 3D structure analysis of the complex formed by Fusarium moniliforme PG and Phaseolus vulgaris PGIP highlighted the role of some active site-residues in the recognition of the enzyme by the PGIP (Federici et al. 2001). However, it was further shown that the complex formation varies according to PG and that PGIP inhibits the enzyme activity although not covering the active site (Bonivento et al. 2008). The presence of PGIP was demonstrated in many plants including those containing low pectin level such as wheat (Kalunke et al. 2015).

3.2.2  Pectin and Pectate Lyases Pectin Lyases-Like (PLLs), include Pectate Lyases (PL: endo-PLs, EC 4.2.2.2 and exo-PLs, EC 4.2.2.9) and PectiN Lyases (PNL: endo-PNLs, EC 4.2.2.10), and catalyse the degradation of HG through a β-elimination process, thus producing unsaturated oligogalacturonides (OGs) that differ from that generated by PG. In the CAZy database, they belong to the PL1, 2 and 3 families. PLLs were identified in plants as well as in bacteria and fungi. A number of studies, in particular on tomato and strawberry, have shown that PLLs play a key role in the regulation of fruit softening (Wang et al. 2018). This was notably demonstrated through the use of transgenic lines silencing or overexpressing fruit-specific PL isoforms, showing that changes in pectin chemistry can affect fruit firmness as well as shelf life and resistance to pathogens (Santiago-Doménech et al. 2008; Posé et al. 2013; Yang et al. 2017). Consequently, the engineering of PLL levels in fruit appears as a strategy of interest to control the quality of the end product. Until now, the determination of the roles of PLL in controlling plant development has remained scarce although a relatively large number of genes encoding PLL are expressed in several plant species,

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including Arabidopsis (Sun and Van Nocker 2010; Cao 2012; McCarthy et al. 2014). Considering the diversity of the expression patterns of PLL, the enzymes are likely to be involved in a number of developmental processes including, for instance, cell separation. A number of PLL from various sources (mainly bacterial and fungal ones) were biochemically characterised, which showed that PL and PNL differ with regards to their substrate specificity, pH-dependence and affinity for cations (Mayans et al. 1997; Herron et al. 2000). Pectate lyases have indeed more affinity for low methyl esterified pectins and an alkaline pH optimum while pectin lyases require high DM pectins and low pH. In contrast, the biochemical characterisation of plant PLLs has proven to be more difficult. However, PLLs from Hevea brasiliensis, Zinnia elegans and mango fruit expressed in heterologous system show optimal activities at rather neutral to alkaline pH (Domingo et al. 1998; Chourasia et al. 2006; Chotigeat et al. 2009). The structure of pectin lyase from Aspergillus niger was determined, showing that the enzyme folds into a parallel β-helix and that it shares structural similarities with pectate lyases albeit low sequence identities (Pickersgill et al. 1994; Mayans et al. 1997). The contribution of pathogen PLLs to virulence and plant infection was recently highlighted (Expert et  al. 2018; Yang et al. 2018; Voxeur et al. 2019). Furthermore, some studies demonstrated the role of plant PLLs in generating resistance to pathogens, likely by the modification of cell wall pectins of the host (Vogel et al. 2002).

3.2.3  Pectin Methylesterases (PMEs) Pectin Methyl Esterases (PMEs, EC 3.1.1.11) belong to the CE8 family of CAZy, and catalyse the specific demethylesterification of methyl ester residues at the C6 of GalA, releasing non-methyl esterified HG, protons and methanol. Non-methyl esterified stretches of HG can be the target of pectin-degrading enzymes such as PG, or cross-linking divalent ions, in particular calcium, to form the so-called egg-box structures. In either case, the PME-mediated changes in pectin structure have dramatic consequences on mechanical properties of plant cell walls (Hocq et al. 2017a). In plants, PMEs are divided into two groups depending on their protein sequence. Group 1 PMEs contain a mature active part while group 2 PMEs have, in addition to the mature part, an N-terminal extension (PRO-region), that can be cleaved by specific subtilisin proteases at dedicated conserved dibasic motifs (RRLL, RKLL…). PMEs are synthesised in the endoplasmic reticulum and the maturation of the protein, including glycosylation and processing of the PRO part, can occur in the Golgi. The processing of group 2 PMEs was reported to play a key role in the export of mature active protein at the cell wall (Wolf et al. 2009). PMEs were shown to control fruit development and fruit ripening, in particular in tomato and in apple (Frenkel et al. 1998; Brummell et al. 2004; De Freitas et al. 2012) but in other species as well (Ross et al. 2011; Wen et al. 2013; Zhang et al. 2019). PMEs play a central role in regulating developmental processes as diverse as primordia emergence at the shoot apical meristem (Peaucelle et al. 2008, 2011), adventitious root formation (Guénin et al. 2011), plant-pathogen interactions (Hewezi et al. 2008; Raiola et al. 2011),

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pollen tube elongation (Jiang et  al. 2005; Leroux et  al. 2015), seed development (Müller et  al. 2013), mucilage production (Turbant et  al. 2016), wood and stem mechanical properties (Siedlecka et al. 2008; Hongo et al. 2012), and dark-grown hypocotyl elongation (Derbyshire et al. 2007; Pelletier et al. 2010; Wolf et al. 2012). Using transgenic lines impaired in the activity of PMEs and multidisciplinary approaches including atomic force microscopy (AFM), it was shown in planta that increased PME activity surprisingly induced a decrease in cell wall stiffness, thus questioning the above-mentioned egg-box model (Peaucelle et  al. 2011; AndresRobin et al. 2018). This highlights the complexity of the regulation of pectin chemistry and could be related to distinct modes of action of plant PMEs and/or the role of pectin degrading enzymes acting synergistically with PMEs (Hocq et al. 2017b). PMEs belong to rather large multigenic families in all plant species sequenced to date (both in monocot and dicot species), which questions the specificity of isoforms and their roles in the context of plant development. PMEs have distinct pH optimum; plant PMEs were reported to have neutral to alkaline optimum while fungal enzymes are more active in the acidic pH range (Jolie et al. 2010; Dixit et al. 2013; Sénéchal et al. 2014). PME activity can be regulated by a number of cations and the pH might influence the mode of action of PMEs, as determined for apple PME (Bordenave 1996; Denès et  al. 2000). The mode of action of fungal and plant PMEs differs. Indeed plant PMEs have been largely described as having a processive mode of action, where the enzyme removes all contiguous methyl esters from a single chain of HG before dissociating from the substrate. In contrast, fungal isoforms are often described as having a random mode of action (Jolie et  al. 2010). These distinct modes of action may have different consequences on pectin structure, which might partially explain the links between pectin chemistry and cell wall mechanical properties in planta. The differences in the mode of action of the PMEs are likely to be related to specific structural features. The 3D-structure was resolved for plant, bacterial, fungal and insect PMEs (Jenkins et al. 2001; Johansson et al. 2002; Di Matteo et al. 2005; Kent et al. 2016). All PMEs share the same distinctive parallel β-helix structure with highly conserved amino acids GxYxE, QAVAL, QDTL, DFIFG, LGRPW, which have been shown to be functionally important (Pelloux et al. 2007). In particular, the active site contains two aspartic acid, two glutamine and an arginine residues and the mechanism of action has been determined: one of the aspartic acid residues, stabilised by the hydrogen bond with arginine, performs the nucleophilic attack on the carboxymethyl group of the HG (Johansson et  al. 2002). Recently, some specific features have been shown to be of importance in determining the processive versus non-processive mode of action, shedding new light on the diversity of these enzymes (Kent et al. 2016). One of the specific features of plant PMEs is their ability to be regulated by proteinaceous inhibitors named Pectin Methylesterase Inhibitors (PMEIs) (Di Matteo et al. 2005). PMEIs are encoded by a large multigenic family, which suggest some specificities in the interaction between isoforms. PME and PMEIs can form a stoichiometric 1:1 complex and the inhibition has been reported to be pH-dependent (Sénéchal et al. 2015). However, some PMEI can inhibit PME activity in a pH-independent manner (Hocq et  al. 2017b) and molecular dynamics (MD) simulation allowed determining the amino acids, which are of importance for the pH-dependence (Sénéchal et al. 2017). Therefore, cell wall

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pH, modulated by PME activity itself, appears as a key element of enzyme activity regulation, presumably through a feedback-loop mechanism (Hocq et al. 2017b). In contrast, pathogen PMEs are not inhibited by any of the PMEI tested so far, and the structural determinants of the lack of inhibition have been also determined (Jolie et al. 2010). A range of chemical compounds has been shown to inhibit either plant or phytopathogen PMEs (Lewis et al. 2008; L’Enfant et al. 2015, 2019). Compared to what has been observed for PG, fungal PME do not appear to be a primary element of the pathogenicity but the plant-PME mediated changes in pectin structure can affect the colonisation of the host plant by fungal or nematode pathogens as well as by viruses (Chen and Citovsky 2003; Lionetti et al. 2007; Hewezi et al. 2008). The PME-mediated production of methanol appears to be of primary importance in the resistance to herbivorous insects acting as a long-­distance signal during plant herbivore interactions (Dorokhov et al. 2012).

3.2.4  Pectin Acetylesterases (PAE) In addition to being methyl esterified, pectins can be acetylated, the degree of acetylation varying from 5 to 30% depending on the species and organs considered. Pectin acetyl esterases (PAEs, EC 3.1.1.6) cleave the acetyl ester bond at O-2 or O-3 positions of GalA in both HG and RG-I (Williamson 1991). The control of the degree of acetylation of pectin by PAE affects the physico-chemical properties of pectins, and is of importance for the activity of other pectin-remodelling enzymes. For instance, the activity of PAE is higher if the pectins have been previously demethyl esterified. PAE have been identified in a wide range of organisms, including plants, fungi and bacteria, but the full biochemical characterisation of plant PAEs have, until now, been impaired by the lack of purified enzymes. Nevertheless, purified plant PAE from mung bean and orange show optimal activity for pH in the range of 5.5 to 6.5. A PAE from Dickeya dadantii, a phytopathogenic bacterium was active on both synthetic substrates and highly acetylated sugar beet pectin (Shevchik and Hugouvieux-Cotte-Pattat 2003). However, most of the biochemical characterization of PAEs used synthetic substrates such as triacetin and p-nitrophenyl acetate, which questions the specificity of the enzyme towards pectic substrates in planta. PAEs belong to the CE13 family of the CAZy database, which is characterised by the presence of the conserved residues serine (S), glycine (G), asparagine (N) and histidine (H), where S and H, together with an aspartic acid (D) form the catalytic triad (Alalouf et al. 2011; Oh et al. 2016). Considering this, it is refereed as belonging to the SGNH protein family. Although until now no 3D crystallographic structure was resolved for PAEs, homology modelling showed that PAEs share structural similarities with human notum that has a palmitoleoyl-protein carboxylesterase activity (Philippe et al. 2017). However, the structural specificities of pathogen and plant PAEs have yet to be determined, notably to understand the biochemical specificity of PAEs as compared to PME, but also compared to rhamnogalacturonan acetylesterases. As other pectin remodelling enzymes, PAEs are encoded by a multigenic family in plants albeit to a smaller extent. Studies related to the roles of

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PAEs in the regulation of pectin structure and plant development are relatively scarce. The contribution of two Arabidopsis PAEs to inflorescence growth (de Souza and Pauly 2015) and the role of pectin deacetylation in pollen tube elongation and reproductive process in tobacco (Gou et  al. 2012) have been revealed using mutants. A link has been further established between PAE-mediated deacetylation and the mechanical properties of potato tuber (Orfila et  al. 2012). Although not formally demonstrated using functional genomics approaches, PAEs are likely to be involved in the control of cell wall structure during pathogen infection as a number of genes encoding PAEs has shown an increased expression following pathogen attack (Philippe et al. 2017).

3.3  Rhamnogalacturonan-Degrading Enzymes 3.3.1  Rhamnogalacturonan-Hydrolases The backbone of rhamnogalacturonan I (RG-I) is made of repeating disaccharide [→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→] units (see Chap. 2). Splitting these two types of linkages involves hydrolases/lyases and endo/exo enzymes (Table  3.1, Fig.  3.1b). The first reported RG-hydrolase (EC 3.2.1.171) was purified from Aspergillus aculeatus and hydrolysed the α-D-GalpA-(1→2)-α-L-Rhap glycosidic bond with an endo mode of action, to release oligosaccharides with a reducing β-D-­ GalpA (Schols et al. 1990). Several similar enzymes were subsequently reported in various strains (Kofod et al. 1994; Sakamoto et al. 1994; Suykerbuyk et al. 1997). To date, Aspergillus genus is recognised as the main producer of rhamnogalacturonan-­ degrading enzymes. Only three enzymes are reported from other sources: Botrytis cinerea (Chen et  al. 1997), Irpex lacteus (Normand et  al. 2010) and Penicillium chrysogenum (Matsumoto et al. 2017). The mode of action of rhamnogalacturonan-­ hydrolase has been further studied. It requires five repeating disaccharides to bind to its substrate (Mutter et al. 1998a) and was hindered by methyl and acetyl esters on the backbone (Schols et al. 1990; Kofod et al. 1994). Conversely, the enzyme from I. lacteus was highly tolerant to acetyl groups (Normand et al. 2010). P. chrysogenum RGH activity was hindered by the presence of galactose on the backbone (Matsumoto et al. 2017). The first solved 3D crystallographic structure of a GH28 enzyme was the RG-hydrolase from Aspergillus aculeatus (Petersen et al. 1997). It forms a right helix of 13 turns of beta strands. Its active site is located in a groove almost perpendicular to the helix axis. It is N- and O-glycosylated at 2 and 18 sites, respectively, for a total of 6 kDa. The exo-hydrolases active on RG-I are of two types: (a) the rhamnogalacturonan galacturonohydrolases (EC 3.2.1.173, GH28) allow the exo-hydrolysis of the α-D-­ GalA-(1→2)-α-L-Rha bond releasing single D-galacturonic acid from the non-­ reducing end of rhamnogalacturonan oligosaccharides (Mutter et al. 1998b), and (b) the rhamnogalacturonan rhamnohydrolases (EC 3.2.1.174, GH78 and 106) allow the exo-hydrolysis of the α-L-Rha-(1→4)-α-D-GalA bond releasing single β-L-­ rhamnose from the non-reducing end of rhamnogalacturonan oligosaccharides

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(Mutter et al. 1994). The former is usually more active on small RG-I substrates whereas the latter is generally hindered by a galactose substitution on the terminal rhamnose (Mutter et  al. 1998b). All the enzymes gathered in GH78 and GH106 release rhamnose from different substrates. They differ in their 3D structures, which have been resolved for α-L-rhamnosidases of each family. The first crystal structure of a GH78 member has been determined for Bacillus sp. GL1 α-L-rhamnosidase B (Cui et al. 2007). It is a five-module structure in which the catalytic one is an (α/α)6-­ barrel. The rhamnosidase from Bacteroides thetaiotaomicron is the only 3D structure resolved in GH106. It follows a (β/α)8-barrel domain with two additional β-strands and it needs a calcium ion to bind the substrate (Ndeh et al. 2017). The most complicated structure of rhamnogalacturonan II (RG-II) is recognised and degraded by the bacteria of the digestive tract (Ndeh et al. 2017). In particular, in Bacteroides thetaiotaomicron, different PUL encode numerous enzymes with narrow specificity for each glycosidic linkages present in this polysaccharide, except 2-O-methyl-D-xylose- α1,3-L-fucose.

3.3.2  Rhamnogalacturonan-Lyases Rhamnogalacturonan-lyases allow the eliminative cleavage of L-α-Rhap-(1–4)-α-­ D-­GalpA bonds of RGI domains leaving L-Rhap at the reducing end and 4, 5-­unsaturated D-GalpA at the non-reducing end according to an endo (EC 4.2.2.23) or an exo (EC 4.2.2.24) mechanism. The tetrasaccharide Δ4,5GalA-α-1,2-Rha-­ α-1,4-GalA-α-1,2-Rha where ∆ means that the non-reducing end is unsaturated, is the major limit product of the RG endo-lyases. The disaccharide Δ4,5GalA-α-1,2-­ Rha can be further split by unsaturated rhamnogalacturonyl hydrolase (EC 3.2.1.172, GH105). Very few RG-lyases are reported in microorganisms (Azadi et al. 1995; Mutter et al. 1996; Pages et al. 2003), as well as in plants (carrot, Stratilova et al. 1998; cotton seed, Naran et al. 2007). Occurrence and roles of RG-lyases in plants have been recently reviewed (Ochoa-Jiménez et  al. 2018). It shows that plant RG-lyases have only around 25% identity with the enzyme from Aspergillus aculeatus and that many of them contain a carbohydrate binding module at the C-terminal side. The endo RG-lyases are grouped in three different polysaccharide-­ lyase (PL) families, PL4, PL9 and PL11. PL4 is a monospecific family containing only RG-endolyases. It is divided in five sub-classes, PL4-1 and PL4-4 contain mainly bacterial enzymes, PL4-2 contains exclusively enzymes from plant origin, and PL4-3 and PL4-5 contain exclusively enzymes from Eukariota. The enzymes in PL9 are almost exclusively from bacterial origin. This family is divided into four sub-classes and contains only two RG-lyases, both from Bacteroides thetaiotaomicron in sub-class PL9-1. The 3D structure of one of these two has been shown to have the same lysine residue and calcium ion in the active site, as in the pectate lyase from the bacterium Dickeya dadantii (Luis et al. 2018). PL11contains endoand exo-lyases in the two sub-classes, only from bacterial origin except two fungal enzymes in sub-class PL11-2 (Aspergillus nidulans and Neocallimastix frontalis),

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showing again the versatility of the genus Aspergillus for the pectin degrading enzymes. Apart from the PL9 family, exo-RG-lyases are also found in the PL26 family. It is a monospecific family containing only exo-RG-lyases, essentially from bacterial origin but the only one for which the activity has been experimentally demonstrated is from Penicillium chrysogenum (Iwai et  al. 2015). The enzyme mainly released unsaturated galacturonosyl rhamnose disaccharide. It bypassed the Gal side chains of RG backbone but did not require Gal decoration for degradation. The enzyme is active in the dimeric form and on the reducing end of RG (Kunishige et al. 2018). Each unit is composed of three domains. Domain III displays a (α/α)6-­ barrel fold and contains the substrate-binding site.

3.3.3  Rhamnogalacturonan-Acetylesterase RG acetylesterases (EC 3.1.1.86, CE 12), required for the deacetylation of the RG-I backbone have been firstly described in Aspergillus species (Searle-van Leeuwen et  al. 1992, 1996; Kauppinen et  al. 1995). As pectins carry acetyl-esters on both homogalacturonan and rhamnogalacturonan (Ralet et al. 2005), it is sometimes difficult to distinguish between HG- and RG-acetylesterase specificity. It needs to have the appropriate substrates corresponding to the two different structural domains (Bonnin et  al. 2008). Only the 3D structure of the Aspergillus aculeatus RG-acetylesterase has been resolved (Mølgaard et al. 2000). The enzyme folds into an α/β/α structure with an open cleft active site.

3.4  Side-Chain Degrading Enzymes Pectin side chains are formed by the neutral sugars arabinose and galactose linked by different linkages depending on whether they belong to type I or type II arabinogalactans (See Chap. 2). The enzymatic degradation of neutral side chains in pectin involves endo- and exo-arabinanases, arabinofuranosidases, endo- and exo-­ galactanases and β-galactosidases (Table 3.1; Fig. 3.1b). As the neutral sugars are sometimes substituted by ferulic acid, the enzymatic degradation also requires feruroyl esterases.

3.4.1  Arabinanases and α-Arabinofuranosidases α-L-arabinofuranose is present in plant cell wall polysaccharides, primarily in pectin arabinan but also in some hemicelluloses (xyloglucan, arabinoxylan). Its release from polysaccharides requires endo- and exo-arabinanases and α-arabinofuranosidase activities. Endo-arabinanases (EC 3.2.1.99, GH43) randomly cleave the α-1,5-­linkages

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in the internal region of the arabinan backbone, preferably on polysaccharides and they are mostly found in microorganisms. In phytopathogenic fungi they have long been shown to play a role in pathogenesis (Stranne and Sakuragi 2016). As an example, in Botrytis cinerea a GH43 endo-arabinanase is a host-specific virulence factor (Nafisi et al. 2014). On the bacteria side, the genus Bacteroides exhibits an arabinan utilization locus with two GH43 arabinanases and two GH51 arabinofuranosidases (Arnal et  al. 2015). So the corresponding encoding sequences are grouped in the genome in a locus dedicated to the arabinan breakdown, together with other sequences dedicated to arabinose binding, transport and utilisation. Plant arabinanases are poorly documented except those from seeds where they would allow the use of arabinan as storage reserve (Gomez et  al. 2009). Exo-arabinanases (no EC number) hydrolyse arabinan from the non-reducing end to release arabinotriose (Kaji and Shimokawa 1984) or arabinobiose (Sakamoto and Thibault 2001). To date they are only described in bacteria and fungi, and belong to the mono-­ specific CAZy family GH93. Arabinofuranosidases (EC 3.2.1.55) release α-L-­arabinofuranose linked to other sugars with (1,2), (1,3) and (1,5) linkages, in arabinan, arabinoxylan, and arabinogalactan. They have been classified according to their substrate specificity and mode of action (Beldman et al. 1997): class A contains arabinofuranosidases not active on polysaccharides, class B contains those active on polysaccharides, and the last class contains those specific for the hemicellulosic arabinoxylan. They are found in several GH families: 43, 51, 54, and 62. This reflects not only differences in their primary sequences and protein structures but also differences in their substrate specificity. Those from GH43 cleave α-(1,5) linkages in linear arabinan and arabino-oligosaccharides while those from GH62 are specific for arabinoxylan. The arabinofuranosidases from GH51 and GH54 have generally broader specificities and can be involved in the hydrolysis of pectin as well as hemicelluloses (Beylot et al. 2001; Yang et al. 2015). Arabinandegrading systems are extensively described in few fungi and bacteria, notably Penicillium purpurogenum (Mardones et  al. 2015; Ravanal and Eyzaguirre 2015; Vilches et  al. 2018), Penicillium chrysogenum (Sakamoto and Thibault 2001; Sakamoto et al. 2011a, 2013a; Shinozaki et al. 2015), and the thermophilic bacterium Caldanaerobius polysaccharolyticus (Wefers et al. 2017). As they are produced by each strain, the three types of enzymes involved in the degradation of arabinan may act synergistically and thus be more efficient, including on branched arabinan. Due to their action towards the different arabinosyl linkages, arabinofuranosidases are key elements in this synergistic process, since they cleave the linkages in the arabinose side chains, allowing arabinanases to cleave the linkages in the backbone.

3.4.2  Galactanases and β-Galactosidases Galactan-degrading enzymes have been reviewed by Sakamoto and Ishimaru (2013). Endo-galactanases include endo-β-1,4-galactanases (EC 3.2.1.89, GH53), endo-β-1,3-galactanases (EC 3.2.1.181, GH16) and endo-β-1,6-galactanases (EC 3.2.1.164, GH5 and GH30). They all randomly cleave the galactan chain in shorter

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oligosaccharides, and endo-β-1,3-galactanases and endo-β-1,6-galactanases are also involved in the hydrolysis of galactose from arabinogalactan-proteins. ­Endo-­β-­1,4-galactanases and endo-β-1,6-galactanases have been described in both fungi and bacteria. In Aspergillus niger, galacturonic acid and arabinose, but not galactose, induce the expression of endo-β-1,4-galactanase (de Vries et al. 2002), suggesting that the gene is co-expressed with genes encoding other pectinolytic enzymes. Another mechanism of expression control seems to predominate in bacteria since galactooligosaccharides induce endo-β-1,4-galactanase expression in Geobacillus stearothermophilus (Tabachnikov and Shoham 2013). To date, two 3D-structures of bacterial endo-β-1,4-galactanases and four 3D structures of fungal endo-β-1,4-galactanases are available (cazy.org/GH53). As expected for enzymes belonging to GH53, their structure reveals a (β/α)(8)-barrel architecture. The enzyme from Bacteriodes thetaiotaomicron presents a relatively open active site cleft compare to that in the enzyme from Bacillus subtilis (Böger et al. 2019). The catalytic residues of the two enzymes align although the enzyme from B. thetaiotaomicron is more active on short oligosaccharides than on polymeric galactan. Endo-­ β-­1,4-galactanases generally accumulate monomers and dimers of galactose. A β-1,3-galactanase acting with an endo mode of action was first evidenced in the winter mushroom Flammulina velutipes (Kotake et  al. 2011). More recently two other endo-β-1,3-galactanases were shown in Aspergillus flavus and in Neurospora crassa (Yoshimi et al. 2017). All three enzymes belong to GH16 family and β-1,3-­ galactotriose has been identified as the smallest possible substrate. The hydrolysis of the galactan structures is further realised by the action of exo-enzymes. Plant, fungi and bacteria produce exo-β-1,4-galactanases (EC 3.2.1.) belonging to GH35 family. Penicillium chrysogenum was the first microorganism reported to produce such an enzyme (Sakamoto et al. 2013b). Its activity was the highest on galactan, decreased with the degree of polymerisation of the substrate and released only galactose. Its synergistic action with the endo-β-1,4-galactanase from the same strain fully degraded lupin galactan into galactose. Due to their specific mode of action, the exo-β-1,4-galactanases from Bacillus subtilis and Aspergillus niger are able to keep the galactose moiety in their active site to add it on an acceptor, thus catalysing a transfer reaction (Nakano et  al. 1991; Bonnin and Thibault 1996). Similarly, the hydrolysis of type II arabinogalactan is realised by the action of exo-­ β-­1,3-galactanases (EC 3.2.1.145, GH43). They break the last linkage on the non-­ reducing end of the polysaccharide even if the last galactose residue is substituted by a side chain (Sakamoto et  al. 2011b). Most of the exo-β-1,3-galactanases described so far from microorganisms contain a CBM at their C-terminus: CBM35 in fungal enzymes from F. oxysporum, I. lacteus, and P. chrysosporium, and CBM13 in bacterial enzymes from C. thermocellum, S. avermitilis, and Streptomyces sp. (Sakamoto and Ishimaru 2013). The difference between exo-galactanases and galactosidases is that the former are highly active on the polymeric galactan while the latter are not. The β-galactosidases (EC 3.2.1.23) belong to several GH. They are mainly grouped in GH1, 2, 35 and 42, but some individuals are also present in GH59, 147 and 165. As already mentioned for arabinofuranosidases, it is some-

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times difficult to know precisely the function of the galactosidases in the hydrolysis of cell wall polysaccharides as galactose is present not only in pectin but also in hemicelluloses (xyloglucan, galactomannan). The β-galactosidase from Bacteroides ovatus is more likely dedicated to pectin degradation as it has been recently shown to belong to a galactan polysaccharide utilizing locus (PUL) (Luis et  al. 2018). However, this enzyme was particularly active on galactan and would therefore be better referenced by the term exo-β-1,4-galactanase. The family GH165 was recently created to classify a new β-galactosidase from the bacterial strain Sinorhizobium meliloti exhibiting low sequence identity to other known GHs (Cheng et al. 2017).

3.4.3  Feruloylesterases Ferulic acid is a hydroxycinnamic acid frequently found in certain pectins. Its release is catalysed by feruloyl esterases, also termed ferulic acid esterases, FAE (EC 3.1.1.73, CE1), which hydrolyse the ester bond between ferulic acid and the carrier sugar. They are produced by many bacteria and fungi. In the CAZy database, they are all members of the carbohydrate esterase family 1 (CE1), and the few crystal structures available to date show the classical eight-strand α/β fold of lipase/ esterase structure. As the carrier sugars and the linkage types are diverse, different classification systems have been proposed. Crepin et al. (2004) proposed to classify FAE into 4 groups (A to D) according to their substrate specificity. The ability to release 5,5′-diferulic acid was also considered (Table 3.2). Interestingly, this classification was supported by amino acid sequence identity. However, this system has been built on a limited number of enzymes and sequences known at that time, and a new sub-class E has been further created to classify the FAEs that do not belong to any of the four previously defined groups. The classification of fungal FAEs has been further refined according to gene sequence (Benoit et al. 2008). The resulting seven sub-families contain FAEs from the three types A, B and C according to Crepin’s classification. The most recent classification of fungal FAEs proposed thirteen sub-families based on both phylogeny and substrate specificity (Dilokpimol et  al. 2016). Only one classification attempted to group FAEs from fungal, bacterial and plant origins (Udatha et  al. 2011). This lead to twelve sub-families based on amino acid composition and secondary structure, but without any biochemical data. However, the high sequence identity between FAEs and other enzymes such as acetyl-esterases or lipases makes this classification without functional data quite inappropriate. The biochemical features of the microbial FAEs reported so far were recently reviewed (Oliveira et al. 2019). Most of them are active at 40–50 °C and pH 6–7. They are most often characterised on synthetic substrate (methylferulate) and this does not allow anticipating easily they action on natural substrates.

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Table 3.2  Classification of feruloyl esterases (adapted from Crepin et al. 2004) Key enzyme

Type A A. niger FaeA

Synthetic substrates MFA, MSA, MpCa Targeted linkages in FA-O-5-Ara natural substrates (xylan) FA-O-6-Gal (pectin) Yes Release of 5-5′ diferulic acid Sequence similarity Lipase

Type B P. funiculosum FaeB N. crassa Fae-1 MFA, MpCA, MCA

Type C A. niger FaeB T. stipitatus FaeC

Type D Piromyces equi EstA

MFA, MSA, MpCA, MCA FA-O-5-Ara FA-O-2-Ara (xylan, pectin)

MFA, MSA, MpCA, MCA

No

No

Yes

CE1 acetyl xylan esterase

Chlorogenate esterase, tannase

Xylanase

3.5  Synergistic Action of Pectin Degrading Enzymes The enzymatic synergy is defined as the cooperation of two or more enzymes to produce a combined reaction greater than the sum of their individual action. Synergistic activities of the numerous hydrolases, lyases and esterases presented above are needed for full degradation of pectin. For this reason, the microorganisms coordinate the production of the different necessary enzymes. As an example, the genome of Aspergillus niger contains twenty one genes encoding GH28 enzymes endo- and exo-PG as well as endo- and exo-rhamnogalacturonases (Martens-­ Uzunova et al. 2006). In Bacteroides species, 30 GHs and PLs are devoted to the degradation of the major pectin domains (Luis et al. 2018). In the PUL unravelled in the Bacteroidetes genome, the physically linked genes are co-expressed and target a unique substrate to improve the efficiency of the degradation (Arnal et  al. 2015; Despres et al. 2016). Similarly in the thermophilic bacteria Caldanaerobius polysaccharolyticus, a gene cluster of six GH has been identified (Wefers et  al. 2017). The enzymes belonged to GH 27, 43, 51 and 127 and were all involved in arabinan degradation. As already mentioned in the previous sections, each activity is produced by a multiple-gene family that results in different proteins with different conditions of action (pH, temperature, recognition site on the substrate etc.). Behind the possible synergy for each strain, this large enzyme set also allows cross-feeding between different species, as demonstrated in the complex microbial community of human gut microbiota (Luis et al. 2018). It is likely that in planta pectin remodelling enzymes can act in a synergistic manner to fine-tune pectin structure and control cell wall mechanical properties that allow growth or differentiation. Although difficult to address in planta, it has been shown that both PME and PG are needed to degrade cell wall in Arabidopsis pollen tetrad, thus allowing proper pollen grain development (Francis et al. 2006; Ogawa et al. 2009).

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3.6  Conclusion To date, the literature about pectin-degrading and modifying enzymes is already very large. However, this is a complex area and there are still enzymes to discover, as some pectin structural motifs are still resistant to the hydrolysis by enzymes discovered so far. This is particularly the case of the RG-II structures. Moreover the growing knowledge about these enzymes allows thinking of a number of applications for the industrial processing of plants, either by using microbial enzymes as technological aids or by controlling the activity of endogenous plant enzymes. Thus, the knowledge of these enzymes paves the way to applications as common as the processing of fruit juices, the extraction of oil or the retting of plant fibres.

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Chapter 4

Isolation and Characterisation of Pectin Gordon A. Morris and Hana A. S. Binhamad

4.1  Introduction There are a large number of fruits and vegetables which have been reported to contain pectins (Table  4.1) including for example, blackcurrants (Alba et  al. 2018), broccoli florets (Houben et al. 2011), carrots (Houben et al. 2011), melon (Denman and Morris 2015), okra pods (Alba et al. 2015; Kpodo et al. 2017), pumpkin (Jun et al. 2006; Košťálová et al. 2010, Košťálová et al. 2013a, b, Košťálová et al. 2014, Košťálová et al. 2016; Simpson and Morris 2014; Sukhenko et al. 2020), sunflower heads (Anger and Berth 1985; Muthusamy et  al. 2019), tomatoes (Round et  al. 1997; Zhang et al. 2019) and several other sources (Ahmadi Gavlighi et al. 2018; Burana-osot et  al. 2010; Cardoso et  al. 2002; Deng et  al. 2019; Grønhaug et  al. 2010; Happi Emaga et al. 2008; Huang et al. 2011. 2016; Inngjerdingen et al. 2007, 2008; Jacobo-Valenzuela et al. 2011; Kazemi et al. 2019a, b; Khodaei and Karboune 2013; Kliemann et al. 2009; Koubala et al. 2008; Kratchanova et al. 1991; Levigne et al. 2002a, b; Liang et al. 2012; Manrique and Lajolo 2002; Meersman et al. 2017; Mikshina et  al. 2015; Minjares-Fuentes et  al. 2014; Muthukumaran et  al. 2017; Ognyanov et al. 2016; Oliveira et al. 2016; Pagán and Ibarz 1999; Pagán et al. 1999; Pereira et al. 2016; Robert et al. 2008; Rojas et al. 2015; Sabater et al. 2020; Sari et al. 2018; Seixas et al. 2014; Sun et al. 2019; Urias-­Orona et al. 2010; Wang et al. 2016; Yapo 2009a, b; Zarei et al. 2017; Zhang et al. 2020). However, many of these “pectins” from novel sources contain large amounts of neutral sugars (e.g., arabinose, galactose and rhamnose) and therefore often contain less than the legal minimum requirement of 65% galacturonic acid and cannot be defined as “pectin” for food and pharmaceutical applications (May 1990). This therefore limits the potential commercial sources of food and pharmaceutical grade pectins (May 1990) and at the present time 85% of all commercial pectins are G. A. Morris (*) · H. A. S. Binhamad Department of Chemical Sciences, University of Huddersfield, Huddersfield, UK e-mail: [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_4

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Table 4.1  Some examples of pectin sources extraction techniques and characterisation methods discussed in this chapter Characteristic sources of pectin Extraction methods Apple pomace Solvent extraction Banana peel Microwave assisted extraction Carrot waste Ultrasound assisted extraction Citrus fruits (orange, Enzyme assisted lemon, grapefruit etc.) extraction Mango peel Supercritical fluid extraction Melon Electromagnetic induction heating extraction Okra pods Subcritical water extraction Passion fruit peel Peach pomace Pumpkin waste Soy beans Sugar beet pulp Tomato waste Watermelon rinds

Characterisation methods Phenol sulfuric acid assay (Dubois) m-hydroxydiphenyl assay Equivalent weight Titrations Infrared (IR) spectroscopy High performance anion exchange chromatography coupled to pulsed amperometric detector (HPAEC-PAD) Nuclear magnetic resonance (NMR) Gas chromatography coupled to mass spectrometry (GC-MS) Molar mass and molar mass distribution Intrinsic viscosity ζ-potential Rheology Capillary zone electrophoresis (CZE) Atomic force microscopy (AFM)

extracted from citrus peels, 14% from apple pomace and 1% from sugar beet pulp (Ciriminna et al. 2016) (see Fig. 4.1). It is also important to note that yield, chemical composition and molecular weight for example will depend on a number of variables including genetic variety (Kpodo et al. 2017), ripening stage (Deng et al. 2019; Prasanna et al. 2007; Seymour et al. 1987) and extraction techniques.

4.2  Extraction of Pectin There are several methods which have been used for the extraction of pectins including, acid extraction, isolation with enzymes (e.g., cellulases, hemicellulases, polygalacturonases), electromagnetic induction heating, microwave and ultrasound associated extraction (Adetunji et al. 2017; Bagherian et al. 2011; Kashyap et al. 2001; Mishra et al. 2012; Wang et al. 2007; Vasco-Correa and Zapata Zapata 2017; Marić et al. 2018; Rodsamran and Sothornvit 2019).

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Fig. 4.1  Major commercial sources of pectin (adapted from (Ciriminna et al. 2016). Lemon peel (48%), lime peel (26%), apple pomace (14%), orange peel (11%) and sugar beet pulp (1%)

4.2.1  Conventional Extraction Methods Extraction of pectin is a process which involves the physical-chemical hydrolysis and extraction of pectin macromolecules from plant tissues (Seixas et  al. 2014) under the influence of different factors, including temperature, pH and extraction time (Adetunji et al. 2017; Pagán et al. 2001). The first step being solubilisation of the protopectin followed by a second hydrolysis reaction where the pectin is degraded, lowering the yield at longer extraction times, and once all the “extractable” pectin has been exhausted the hydrolysis predominates (Pagán and Ibarz 1999; Pagán et  al. 1999). Therefore, the temperature, pH, duration of extraction, type of acid, number of extraction cycles, the ratio of water to raw material and volumes of organic solvent can modify the quantity as well as the quality of the extracted pectins (Denman and Morris 2015; Kliemann et al. 2009; Kumar et al. 2010; Levigne et  al. 2002a; Pagán and Ibarz 1999; Pagán et  al. 1999; Samavati 2013; Sudhakar and Maini 2000). In general, the highest yields of pectins are obtained by the most suitable method for industrial extraction of pectin which is hot acid (pH 1.5–2.5) extractions at temperatures around 70–90 °C (May 1990). Acid extraction has another advantage which is that the pectin obtained is ordinarily enriched with galacturonic acid residues. This however can result in pectin degradation (Pagán and Ibarz 1999; Pagán et al. 1999) and environmental issues surrounding the disposal of hazardous chemicals (Minjares-Fuentes et al. 2014) which has driven a large amount of research in alternative non-conventional extraction techniques (Adetunji et al. 2017; Bagherian et al. 2011; Fishman et al. 2006; Košťálová et al. 2016; Minjares-Fuentes et al. 2014; Qiu et al. 2019; Rodsamran and Sothornvit 2019; Rojas et al. 2015; Sabater et al. 2020; Sari et al. 2018; Seixas et al. 2014; Vasco-Correa and Zapata Zapata 2017; Wang et al. 2007, 2016; Zarei et al. 2017; Marić et al. 2018). The isolation of pectins has also be carried out on a laboratory scale using of chelating agents (e.g., potassium oxalate), dilute sodium hydroxide

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solutions, water (hot or cold) and buffered solutions (Alba 2015). The extraction with chelating agents has a disadvantage in that it is difficult to remove the residual chelates, also alkaline extraction facilitates reduction of chain length and in the degree of acetylation and methylation by β-elimination (Rombouts and Thibault 1986).

4.2.2  Enzymatic Extraction Enzymes can be used during pectin extraction to hydrolyse the matrix of the plant cell wall and therefore improve the permeability of the plant cell wall to solvent and improve mass transfer. The most commonly enzymes are cellulases, proteases, hemicellulases and xylanases or commercial formulations which contain one or more of these enzymes for example Celluclast®, Econase®, Neutrase® or Viscoferm® (Adetunji et al. 2017; Marić et al. 2018; Wikiera et al. 2015; Panouillé et al. 2006). The yield of pectin will depend on extraction time, enzyme activity, temperature and pH and therefore extraction conditions need to be optimised for specific enzymes (Adetunji et  al. 2017; Marić et  al. 2018) and some “typical” extraction conditions have been outlined previously (Adetunji et al. 2017; Marić et al. 2018; Wikiera et al. 2015). Enzyme assisted extraction usually requires lower temperatures and less solvents than conventional acid extraction, however there are disadvantages in terms of the high cost of enzymes and issues around scale-up (Adetunji et al. 2017; Marić et al. 2018).

4.2.3  Microwave-Assisted Extraction The exposure of a sample matrix and/or solvent to microwave energy causes a disorganised rotation of any polar molecules which results in heat (Adetunji et  al. 2017; Bagherian et  al. 2011; Marić et  al. 2018), as with other non-conventional pectin extraction technologies this can reduce extraction times and solvent usage (Adetunji et al. 2017; Marić et al. 2018). There have been a number of studies on the extraction of pectins from a number of sources (Adetunji et  al. 2017; Bagherian et al. 2011; Fishman et al. 2000, 2006; Kazemi et al. 2019a; Košťálová et al. 2016; Marić et  al. 2018; Rodsamran and Sothornvit 2019; Rojas et  al. 2015; Samavati 2013; Sari et  al. 2018; Seixas et  al. 2014; Wang et  al. 2007; Zarei et  al. 2017). Experimental conditions usually require the optimisation of microwave assisted extraction conditions (power/temperature, time and solid: liquid ratio). However as with acid extraction prolonged extraction under “harsh” conditions may degrade the pectin chain (Fishman et al. 2006). For more information see for example (Adetunji et al. 2017; Marić et al. 2018).

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4.2.4  Ultrasonic Extraction Ultrasonic waves travelling through a liquid medium, for example acidified (pH 1.5) water (Bagherian et al. 2011), causes bubbles/cavities to form, expand and collapse in a process called cavitation (Adetunji et al. 2017; Marić et al. 2018). This process occurs over a very short time period (~400 μs) and can lead to very high temperatures and pressures which will enhance solubility in the solvent and increase the rate of mass transfer (Adetunji et al. 2017; Marić et al. 2018). Rather than a conventional ultrasonic bath pectin extractions are usually investigated using an ultrasonic probe, as this results in more efficient cavitation (Adetunji et  al. 2017) and there have recently been a number of ultrasound assisted extractions of pectins form different sources (Bagherian et al. 2011; Košťálová et al. 2010; Minjares-Fuentes et al. 2014; Sabater et al. 2020; Wang et al. 2016). Like acid and microwave assisted extraction, experimental conditions (power/temperature, time and solid: liquid ratio) are important in terms of optimisation (Adetunji et al. 2017). As with other extraction/solubilisation methodologies longer extraction times under more “harsh” conditions have been reported to increase yield whilst decreasing polymer chain lengths (Bagherian et al. 2011; Kök et al. 2009; Mohod and Gogate 2011).

4.2.5  S  ub-Critical Water Extraction/Accelerated Solvent Extraction Sub-critical water extraction uses liquid water at temperatures above its usual boiling point without a change in phase, this method is also called pressurised hot water extraction or superheated water extraction or more generally accelerated solvent extraction. The use of conventional solvents and elevated temperatures and pressures which facilitates the penetration of the solvent in to the sample matrix, increases solubility and mass transfer whilst reducing solvent viscosity and surface tension. Again the yield and quality of the pectin is correlated with the extraction conditions (pressure, temperature, time and solid: liquid ratio) and optimisation is required (Chen et al. 2015; Le Normand et al. 2011). In this section some of the methods of pectin extraction have been discussed, but it is important to note that these extraction techniques could be used sequentially for example ultrasonic extraction prior to microwave assisted extraction (Bagherian et al. 2011) or ultrasonic extraction combined with subcritical water extraction (Chen et al. 2015). After extraction, the crude pectin extracts are then separated by a filtration, centrifugation, precipitation with alcohol (methanol, ethanol or isopropanol) or by precipitation with an insoluble salt e.g., aluminium (May 1990). The precipitate obtained is washed with alcohol and pressed to remove soluble contaminants, and finally dried and milled to get powdered pectin (Joye and Luzio 2000).

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4.3  Characterisation of Pectin After extraction and isolation, it is essential to fully characterise pectin polymers. This section describes current methods widely used in the physico-chemical characterisation of pectins.

4.3.1  Phenol-Sulfuric Acid/Total Carbohydrate Assay Upon the addition of sulphuric acid, pectins when subjected to heat will hydrolyse and form furan derivatives which may further react with a phenol solution to produce a coloured complex (λmax ~ 490 nm), although different monosaccharides react differently and uronic acids, such as galacturonic acid have a relatively low colour response (Dubois et al. 1956). The intensity of the colour produced will depend on the amount of pectin. However, it is important to note that this is a relative technique and the quality of the results will depend the reaction time and the choice of an appropriate standard i.e., galacturonic acid for pectin analysis.

4.3.2  G  alacturonic Acid Content and Degree of Methyl Esterification (DM) 4.3.2.1  m-Hydroxydiphenyl Assay Upon the addition of sulphuric acid, pectins when subjected to heat will hydrolyse and form furan derivatives which may further react with a m-hydroxdiphenyl solution to produce a pink complex (λmax ~ 520 nm). The galacturonic acid content can then be determined from a calibration curve of galacturonic acids standards typically over the concentration range 10–100  μg  mL−1 (Brummer and Cui 2016; Filisetti-Cozzi and Carpita 1991). This method is less sensitive to the presence of other monosaccharides or protein molecules than earlier methods (Brummer and Cui 2016). 4.3.2.2  Equivalent Weight A pectin sample (0.5% w/v) is dissolved in a 5% v/v ethanol solution and the acidic solution is titrated with sodium hydroxide (0.1N) in the presence of an appropriate indicator e.g., methyl red (Alfa Ezhil Rose and Abilasha 2016) while sodium chloride can be added to sharpen the end point (Owens et al. 1952). This titration can be used to give an estimate of total anhydrouronic acid content and the neutralised solution can then be used to estimate degree of methyl esterification (Owens et al. 1952).

4  Isolation and Characterisation of Pectin



Equivalent weight =

67

1000 × Weight of sample ( g )

Volume of NaOH ( mL ) × Normality of NaOH

(4.1)

After having neutralised the free galacturonic acid the degree of methyl esterification (methoxyl content) can determined from a further titration. The pectin is saponified by the addition of 25 mL of 0.25N sodium hydroxide solution in left at room temperature for 30 min after which time 25 mL of 0.25N hydrochloric acid is added and the excess acid is back titrated with 0.1N sodium hydroxide as per the equivalent weight titration described previously. From which total anhydrouronic acid content (AUA) and %DM can be calculated (see Eqs. (4.2), (4.3), and (4.4)): Methoxyl content ( %MeO ) =

Volume of NaOH ( mL ) × Normality of NaOH × 3.1 Weight of sample ( g )



%AUA = +



(4.2)

Volume of NaOH ( mL ) from Eq. (1) × Normality of NaOH × 100 × 176 Weight of sample ( g ) × 1000

Volume of NaOH ( mL ) from Eq. ( 2 ) × Normality of NaOH × 100 × 176 Weight of sample ( g ) × 1000

%DM =

(4.3)

176 × %MeO × 100 (4.4) 31 × %AUA

where 176 and 31 are the formula weights of AUA and MeO, respectively. 4.3.2.3  Infra-Red (IR) Spectroscopy The degree of methyl esterification can also be determined directly from infra-red spectroscopy (IR) (Chatjigakis et  al. 1998; Filippov and Kohn 1975; Gnanasambandam and Proctor 2000; Manrique and Lajolo 2002; Chylińska et al. 2016). This can be done quite simply by measuring the absorbance of the stretching vibrations for a free carboxylic acid group (COO-) at approximately 1630 cm−1 and of the corresponding methylated group (COOMe) at approximately 1740  cm−1 (Chatjigakis et al. 1998; Filippov and Kohn 1975; Gnanasambandam and Proctor 2000; Manrique and Lajolo 2002; Szymańska-Chargot et al. 2016). This is usually done by plotting the calibration curve for pectins of known degree of esterification versus the area (Chatjigakis et al. 1998; Manrique and Lajolo 2002) using the following equation:

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G. A. Morris and H. A. S. Binhamad



DM% =

( Area of COOMe peak ) (4.5) ( Area of COOMe peak ) + ( Area of COO− peak )

4.3.3  Sugar Composition 4.3.3.1  H  igh Performance Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD) This method allows the direct determination of pectic monosaccharides without any need for prior derivatisation. In brief, pectin samples are hydrolysed to their monosaccharide constituent sugars using a strong acid usually trifluoroacetic acid (TFA) as this is easier to remove by evaporation. The monosaccharides are then separated using a high performance anion exchange column (Corradini et al. 2012) at a pH of ~12 using for example 10 mM sodium hydroxide, for the separation of uronic acids sodium acetate is often added to the mobile phase to increase the ionic strength and decrease their retention time. This high pH will impart either a fully or partially ionised negative charge on the individual monosaccharides and they can be separated depending their respective acid dissociation constants, pKa (Table 4.2). The column effluent is then detected using a pulsed amperometric detector (PAD), however the detector response is not equivalent for the different monosaccharides, therefore calibration curves generated from purified monosaccharide standards are required for quantification (Brummer and Cui 2016). 4.3.3.2  Nuclear Magnetic Resonance (NMR) NMR is powerful technique in the analysis of pectins and allows detailed structural information (Cheng and Neiss 2012). This technique is based on the net spin of atomic nuclei in an applied magnetic field and nuclei with nuclear spins of 1/2 (e.g., 1 H and 13C) are widely used in the analysis of pectins. The magnetic field experienced by the nucleus is not equal to the applied magnetic field due to shielding from electrons and this results in a chemical shift (relative to a reference). This chemical shift Table 4.2  pKa values for typical pectic monosaccharides at 25 °C adapted from (Bhattacharyya and Rohrer 2012; Kaijanen et al. 2015)

Monosaccharide Glucose Galactose Xylose Mannose Rhamnose Arabinose Galacturonic acid

pKa 12.28–12.35 12.39 12.15 12.08 11.84 12.34–12.43 3.48

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Fig. 4.2 Typical 1H NMR spectrum for okra pectin in D2O at 70 °C where some of the peaks have assignments have been identified. A methylated galacturonic acid residue is shown for clarification (Binhamad 2018)

is a function of the nucleus and its local environment (Cheng and Neiss 2012) and can be used to identify specific protons on each monosaccharide unit (Fig. 4.2). Pectins are usually dissolved in a deuterated solvent, for example D2O to allow the –OH groups to be fully deuterium exchanged (Cheng and Neiss 2012). This may require the pectin being dissolved in D2O then freeze-dried prior to re-dissolution in D2O with this process being repeated on several occasions. Furthermore, due to high molecular weight/ viscosity of pectin solutions it is often advisable to run pectin samples at higher temperatures and/or higher magnetic field strengths for greater spectral resolution. Integration of the acetic and methanol peaks at ~1.92 and 3.36 ppm after saponification can also be used to quantify the degree of methylation and acetylation (Müller-Maatsch et al. 2016). Further detailed structural information can be obtained from 2D-NMR methods for example correlated spectroscopy (COSY), correlated spectroscopy (TOCSY), heteronuclear single quantum correlated spectroscopy (HSQC) and heteronuclear multiple quantum correlated spectroscopy (HMBC). 4.3.3.3  Gas Chromatography Coupled to Mass Spectrometry (GC-MS) Gas chromatography coupled to mass spectrometry is often used in pectin analysis as it is one of the few techniques which has the capability of identifying the linkages which form the polysaccharide chain (Carpita and Shea 1989; Sparkman et  al. 2011). GC-MS is often the preferred method of analysis as it requires less material than other techniques e.g., NMR (Lindhorst 2000). In order to prepare the sample

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for linkage analysis, methylation and acetylation are required to identify where the constituent monosaccharides are linked to one another via glycosidic bonds. This analysis is done by methylating all the free –OH groups in the sample and then using acid hydrolysis to break the glycosidic bonds. Partially methylated alditol acetates (PMAAs) are then produced by reducing and acetylating the methylated sugars. The positions of these groups on the individual monosaccharide can be determined using GC-MS and it is therefore possible to identify the types of links in the chain i.e., it will be possible to tell if a hexose is 1–4 linked or an end terminal etc. (Sims et al. 2018). For molecules containing uronic acids the uronic acid residues can be reduced using sodium borodeuteride (NaBD4) prior to methylated, hydrolysis and acetylation (for more details see (Sims et al. 2018)).

4.3.4  Molar Mass and Molar Mass Distribution One of the most important parameters when characterising pectins (or any other polymers) is the molar mass or molecular weight, expressed in g mol−1 or Daltons (Da). Pectins, like other polysaccharides, are poly(disperse) and it is therefore important to consider the molar mass distribution (Fujita 1962) and the molar mass “averages”: Mn (number average), Mη (viscosity average), Mw (weight average) and Mz (z-average), where for heterogeneous systems Mn < Mη~Mw < Mz. The type of average calculated will depend on the type of instrumentation method used. Number average molecular weight is calculated from end group analysis and osmotic pressure (Devine 1974; Harding et al. 1991b) and are limited in their applicability to high molecular weight polyelectrolyte molecules such as pectin and have been to some extent been superseded by other techniques (Harding et al. 1991b). Viscosity average molecular weight is calculated from intrinsic viscosity measurements (see the section on intrinsic viscosity for more details) and usually requires some knowledge or assumptions about the dilute solution conformation of the pectin. In the last 40  years size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) has become the method of choice in the determination of the molecular weight of pectins (Harding et al. 2016; Morris et al. 2014). In brief, size exclusion chromatography is based on the separation of molecules due to their hydrodynamic radius using a chromatographic column consisting of a matrix of porous polymer beads. Pectin molecules depending on their size will partition in and out of these pores. The partition between pores can be described using the distribution coefficient, KD (0 ≤ KD ≤ 1). Therefore, the total volume accessible to the pectin molecules is the retention volume VR. If KD = 0, then VR = Vo and the molecule is too large to diffuse into the column matrix, this is known as the void volume, and when KD = 1 the polymer can penetrate in to the entire bead matrix and VR = VT, which is called total volume. However, the major disadvantage of a standalone SEC system is that one can only assign relative molecular weights through calibration with standards of known molecular weight which relies on both the standards and sample of interest behaving at least similarly in the SEC columns. However abso-

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71

lute estimates of molecular weight can be calculated with the appropriate detection system for example multi-angle light scattering (MALS). Light scattering enables the absolute determination of molecular weight for pectin molecules. Pectin molecules are often of relatively high molecular weight (>100,000 g mol−1) and therefore a single molecular can have several scattering points and this reduces the scattering intensity at all angles other than zero. Therefore, scattering is usually measured at several angles (up to 18) and extrapolated to zero angle (Debye 1944; Wyatt 1993; Zimm 1948). Therefore, with the addition of a suitable on-line concentration detector, usually refractive index for pectins, one can calculate the molecular weight distribution and the radius of gyration distribution without any prior assumptions of pectin conformation (Tanford 1961). The MALS detector can also be coupled to asymmetric flow field-flow fractionation (AF4-MALS) which can also be used in the molecular weight determination of pectins (Thielking and Kulicke 1998). Asymmetric flow field-flow fractionation (AF4) separates pectin molecules based on their differences in the diffusion coefficient, which is related to their size and shape (Nilsson et al. 1996). AF4 is a non-chromatographic separation technique and separation is achieved by flow through a channel to which a perpendicular force is applied (Fig. 4.3). The channel consists of an upper solid plate which is imperme-

Fig. 4.3  Schematic representation of an asymmetric flow field flow fractionation system where pectin molecules of smaller (filled green circle) hydrodynamic volumes (larger diffusion coefficient) are experiencing a greater flow velocity than those with intermediate diffusion coefficients (filled brown circle) and larger (open circle) hydrodynamic volumes (smaller diffusion coefficient)

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able to solvent and a lower (accumulation) plate which is permeable to solvents (Pauck and Cölfen 1998; Wahlund and Giddings 1987). The laminar flow of the mobile phase creates a parabolic flow profile within the channel; therefore, the stream moves more slowly closer to the lower plate wall than it does in the channel centre (Fig. 4.3). Since separation is based on diffusion coefficient, the smaller molecules tends to elute faster than the larger molecules as they are able to diffuse more readily in to the higher flow zones in the channel (Runyon et al. 2014). The use on-­ line MALS and refractive index detectors can then be used to estimate distributions of molecular weight and radius of gyration. Despite the convenience of SEC-MALS and AF4-MALS, there are several limitations. For example, for large molecular weight polysaccharides SEC columns can give poor separation or non-size exclusion interactions with the columns can give anomalous results. Whereas in the case of field-flow fractionation systems problems of non-inertness through anomalous interactions with the membranes can also lead to erroneous results (Harding et al. 2016). The molecular weight of pectins can also be determined using either sedimentation equilibrium in the analytical ultracentrifuge (SE-AUC) and with prior knowledge of pectin conformation using sedimentation velocity in the analytical ultracentrifuge (SV-AUC) (Harding et  al. 1991a, 2011a, b; Morris et  al. 2000, 2014) which offers a complementary orthogonal approach to MALS with inherent fractionation ability without the need for columns or membranes. Although probably being the “gold standard” in molecular weight determination of polymers these methods have not been extensively thus far in the molecular weight determination of pectins and the theory and appropriate methodologies have been described and reviewed previously (see for example, (Devine 1974; Harding 1995a, b, 2005a, b; Harding et al. 2015; Harding et al. 2010, 2011a; Harding et al. 2016; Harding et al. 1991a; Harding et al. 2011b; Morris et al. 2014; Morris et al. 2000).

4.3.5  Intrinsic Viscosity The intrinsic viscosity is an important parameter which gives information about the size and conformation of a pectin polymer in dilute solution. Intrinsic viscosity is usually measured using an Ostwald capillary viscometer, the flow of pectin molecules in solution through the capillary will follow Poiseuille’s law. From this the ratio of viscosities can be calculated—relative viscosity:

 t  ρ  ηrel =     (4.6)  t0   ρ0 

where t is the flow time for the pectin solution, to is the flow time for the solvent (e.g., water). Due to the measurements being commonly made at low concentrations, ρ/ρο can often be ignored (see e.g. (Harding 1997)), although densities of

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pectin solutions can be measured routinely and for analytical centrifugation based techniques are essential. The specific (ηsp), viscosity is defined as follows:

η sp = ηrel −1 (4.7)



and this, divided by concentration, c (g mL−1) is known as the reduced specific viscosity, ηsp/c (mL g−1). To minimise non-ideality effects, measurements that are made at several concentrations are extrapolated to infinite dilution using the Huggins or Kraemer approaches, or both (Huggins 1942; Kraemer 1938):

η sp c

= [η ] (1 + K H [η ] c ) (4.8)

ln (ηrel ) c

= [η ] (1 − K K [η ] c ) (4.9)

where the intrinsic viscosity [η] is taken as the is the mean of the intercepts from Eqs. (4.8) and (4.9) and KH and KK are the Huggins and Kraemer constants respectively. A useful method for measuring intrinsic viscosities is to calculate the relative and specific viscosities at one concentration and utilise the Solomon-Ciutâ or Solomon-Gottesman approximation (Solomon and Ciutǎ 1962; Solomon and Gottesman 1968). The intrinsic viscosity can then be accurately estimated (error generally ~1%) by a single measurement at low concentration (see for example (Morris 2001)).

[η ]

( 2η ≈

− 2 ln (ηrel ) )

1/ 2

sp

c

(4.10)

Differential pressure viscometers (DPV) based on the 4-capilliary bridge design (Haney 1985a, b) can be utilised on-line and integrated with the SEC-MALS system and the specific viscosity can then be calculated as follows:

η sp =

4 ∆P (4.11) Pi − 2 ∆P

where Pi is the inlet pressure and ΔP the differential pressure across the midpoint of the bridge There the intrinsic viscosity can then be estimated as a function of elution volume using the Solomon-Ciutâ (Gotesman) approximation (Eq. (4.9)) where the weight average viscosity across the entire peak area corresponds to the bulk solution intrinsic viscosity measured using a traditional Ostwald viscometer. The intrinsic viscosity can then be related to the molecular weight via the Mark-Houwink power law relationship.

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[η ] = κη M a (4.12)

where κη and α are obtained from the intercept and slope of the double log plot of [η] vs. Mw The value of a can be used as an estimation of gross macromolecular conformation and hence a values of ~0 correspond to spheres, 0.5–0.8 to random coils, and up to 1.8 to rigid rods (see (Harding 1997)). Typically, pectin has a Mark-­ Houwink α value in the range 0.62–0.94 (Anger and Berth 1985; Axelos et al. 1987; Axelos and Thibault 1991; Berth et al. 1977; Fishman et al. 2001, 2006; Garnier et al. 1993; Harding et al. 1991a, b; Malovikova et al. 1993; Masuelli 2011; Morris et al. 2000, 2002, 2008; Qiu et al. 2019), although this will depend on the source, genetic variety and extraction method, etc.

4.3.6  Rheology The physical behaviour (deformation and flow) of substances, is classified in two categories: viscous liquid or elastic solid. Materials, like pectins, which exhibit both elastic and viscous behaviours are referred to viscoelastic materials (Mezger 2006) and these properties are important in terms of their functionality as gelling and thickening agents (May 1990). 4.3.6.1  Viscosity Depending on viscous behaviour of fluids as a function of shear rate and stress, they are categorised as either Newtonian or non-Newtonian systems (Chhabra and Richardson 1999). This relationship is defined according to Newton’s law:

η=

τ (4.13) γ

When temperature is constant for a Newtonian fluid the relationship between the applied shear stress and shear rate is linear and there will be no change in viscosity when shear is applied, for example: water, oils, alcohol (Chhabra and Richardson 1999). In contrast, for non-Newtonian fluids the relationship between shear stress and shear rate is non-linear and viscosity will change when shear is applied and three types of flows are considered: shear-thickening (dilatant), shear-thinning (pseudo-plastic) and Bingham plastic (Chhabra and Richardson 1999). Pectin solutions behave as a Newtonian liquid up to a certain concentration, although this concentration will depend on the physical properties of the pectin (Mw, [η], radius of gyration), above this concentration pectin solutions are shear-thinning and therefore viscosity decreases with shear rate which is important from a processing as well as an organoleptic point of you. Viscosity is usually measured as a function of shear

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rate (e.g., 0.1–1000  s−1) using a rheometer with a cone and plate geometry and extrapolated to zero-shear (ηο) using for example the Cross model (Morris 1990). Furthermore, as the pectin concentration increases the physical entanglement between polymer chains will increase and above a critical concentration (c*) pectin chains can no longer distribute themselves randomly throughout the solution and adjacent chains will interpenetrate. This is called the “semi-dilute” regime and a further transition to the “concentrated” regime occurs at c**. Viscosity and the critical coil overlap concentration (c* and c**) will depend on degree of esterification, solvent environment (i.e., salt concentration, sugar concentration and pH) and temperature (Oakenfull 1991).

4.4  Conclusions Pectins are a family of chemically diverse biopolymers which are used extensively in the food and pharmaceutical industries as for example gelling/thickening agents, emulsifiers or excipients. There are a number of physico-chemical properties which are important in these applications including monosaccharide composition, degree of esterification and molecular weight all which will depend on the sources of pectin and the extraction conditions. In order to extract and characterise a novel pectin there are a number of recommendations which may be of use to a researcher new to the field of pectin chemistry. I would suggest a simple acid based extraction with an experimental design to measure the effects of pH, extraction temperature and time (Denman and Morris 2015; Kumar et al. 2010; Levigne et al. 2002a). There are then a number of important characterisations which would need to be undertaken including galacturonic acid content, degree of methyl esterification and the weight average molecular weight. The determination of galacturonic acid content using the m-hydroxydiphenyl assay is a simple colourimetric method which is easy to perform and gives reliable results even in the presence of interfering protein molecules. Degree of esterification can also be estimated using titrimetric analysis which is simple, robust and available in every chemistry laboratory. Further characterisation with HPAEC, GC-MS and NMR will give much more detailed compositional and structural information. Finally, molecular weight determination is also very important and requires the use of more specialised equipment (SEC-MALS, AF4-MALS or AUC) which may be not be available in a number of laboratories. However, with some knowledge of the pectin conformation, molecular weight can be estimated using a simple capillary viscometer and the Mark-Houwink power law relationship.

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Chapter 5

Emulsification Properties of Pectin Katerina Alba and Vassilis Kontogiorgos

5.1  Introduction Food and pharmaceutical industry frequently design their formulations aiming to improve human health (e.g., foods that lower cholesterol), produce products with consumer-tailored specifications (e.g., products for vegetarians) or deliver bioactives to the required site of uptake (e.g., colon). Among other biopolymers, pectin may be also used as a carrier for the protection and targeted delivery of bioactive compounds and for increasing their shelf life and stability (Rehman et al. 2019). The challenges arise from the increasing public interest in the availability of “natural” food ingredients where only naturally available materials such as carbohydrates or proteins should be used in the formulations. In addition, complexities also arise from the gastric environment that usually the product needs to bypass before reaching the desired location in the gastrointestinal tract. Polysaccharides, in general, are routinely used in food and pharmaceutical industries, mostly as thickeners, dispersion stabilisers or water structuring agents. These functional properties are employed to create structures with reproducible physical properties. In recent years, however, the need to create advanced formulations that bypass gastric environment, delay lipid digestion to prolong satiety, and deliver bioactives in the gastrointestinal tract at the site of interest has boosted research on the fundamental properties of polysaccharides at interfaces (McClements and Jafari 2018; Araiza-Calahorra et al. 2018; Kontogiorgos 2019). The main reason is that polysaccharide-based structures may resist attack from proteases as well as the acidic environment of stomach that frequently impair the performance of protein K. Alba Department of Biological Sciences, University of Huddersfield, Huddersfield, UK V. Kontogiorgos (*) School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD, Australia e-mail: [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_5

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and surfactant-based formulations (McClements and Gumus 2016). In addition, surface active compounds are used in acidic drinks to emulsify flavour oils, prevent their oxidation and deliver them in a sustained manner (e.g., in the oral cavity) (Matalanis et al. 2011). Technological performance of polysaccharides as emulsifiers is controlled by their macromolecular properties (e.g., conformation, surface charge density, molecular weight etc.) and intra- and inter-chain interactions that act cooperatively to determine adsorption strength (Kontogiorgos 2019). Pectin is obtained from natural sources using suitable extraction methodologies and may be tailored with chemical or physical modifications to improve the functionality of the extracted material. Depending on the source of extraction (Chap. 4) pectin has the ability to rapidly adsorb at the interface, reduce interfacial tension to facilitate droplet disruption, and impede droplet aggregation. This is typically attributed to the presence of hydrophobic elements in the structure such as proteins, ferulic acids, methyl or acetyl groups (Alba and Kontogiorgos 2017). The objective of this chapter is to identify the role of the surface active functional groups and provide a mechanistic understanding of the phenomenology of pectin adsorption at the oil-water interface.

5.2  Role of Structural Elements on the Interfacial Activity The emulsifying capacity of pectin is typically associated with the chemical structure of its backbone such as the degree of methylation (DM) and acetylation (DA), the macromolecular characteristics of pectin chains (molecular weight (Mw), branching, hydrodynamic volume etc.) and the presence of functional units such as protein or ferulic acids. The evaluation of the contribution to the emulsification capacity of pectin of each of these structural parameters is still in progress and a matter of debate. However, some general principles may be drawn that may form the basis for further investigations and greater understanding of pectin functionality at the oil-water interface. In this section, we identify the most important structural elements that contribute to its interfacial activity.

5.2.1  The Role of Protein The protein content in pectin varies depending on the source, isolation conditions and detection methods with higher values typically reported for sugar beet (up to ~9%) and okra (~5%), in contrast to citrus or apple pectin (e.g., ~3% and ~1%, respectively) (Funami et al. 2011; Yapo et al. 2007a; Chen et al. 2016a, 2018; Alba et al. 2015; Schmidt et al. 2015). Proteins are either present as contaminants that are co-extracted during the isolation process or associated with pectin structure through covalent linkages usually attached on the side chains. This association has been also probed by atomic force microscopy describing the protein-pectin complexes as

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“tadpoles” or as a network of “rods and spheres” (Fishman et  al. 2015; Kirby et al. 2008). Sugar beet pectin stabilised emulsions require about 3% protein for optimum surface activity (Chen et  al. 2016a, 2018) whereas enzymic removal of protein results in reduction of interfacial activity and increase of droplet size compared to emulsions fabricated with non-enzymically modified pectin (Funami et al. 2007). The enzymatic treatment also reduces its molecular weight and radius of gyration thus restricting its steric stabilisation efficiency. It has been also shown that adsorbed pectin fractions at the oil-water interface have high protein concentration hinting at the importance of the protein component on emulsion stability (Leroux et al. 2003; Akhtar et al. 2002; Yapo et al. 2007a; Siew and Williams 2008b; Nakamura et al. 2004). Some pectins, as for instance those from pomegranate peel, show limited capacity to lower the surface tension, and its emulsifying properties are mostly attributed to the presence of protein and ester groups (Yang et al. 2018). In contrast, protein-rich and protein-depleted sugar beet pectin fractions have shown a range of emulsion stabilisation properties with protein playing a secondary role (Karnik and Wicker 2018; Chen et al. 2018). Further complications may also arise from the fact that in some pectins covalently-linked ferulic acid-arabinogalactan-protein complex has more notable impact on the interfacial activity and emulsifying capacity than protein alone (Chen et al. 2016b, 2019; Siew and Williams 2008b). Another school of thought proposes that the accessibility and chemical nature of protein (e.g., amino acid composition and conformation) is more important determinant of emulsification capacity than its overall concentration. For instance, sugar beet pectin fractions with different protein amount ranging between 0.8% and 5.9% result in formation of emulsions of comparable droplet sizes and stability (Williams et  al. 2005). In addition, extensin, a hydroxyproline-rich glycoprotein associated with the plant cell walls, was reported to be the main protein-type in pectin isolated from a range of botanical sources (Karnik et al. 2016; Nuñez et al. 2009). However, similar to total protein content, hydroxyproline-rich fractions did not show good emulsifying capacity and could not be directly associated with the emulsifying activity of sugar beet pectin. This is in general agreement with other investigations that have not identified a direct relationship between protein content and emulsifying capacity (Yapo et al. 2007a; Alba et al. 2016) suggesting that protein accessibility to the interface may be hindered by the bulky carbohydrate chains thus restricting interfacial arrangement (Castellani et  al. 2010). A mechanistic description of the complex relationships between protein and pectin at the interface is presented in Sect. 6.3 where the different modes of adsorption are detailed.

5.2.2  The Role of Acetyl and Methyl Groups Acetyl groups, similarly to ferulic groups, enhance interfacial activity of pectin resulting in smaller droplets during emulsification (Akhtar et  al. 2002; Dea and Madden 1986; Leroux et al. 2003; Siew and Williams 2008a). Early studies using

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de-acetylated pectin revealed that the presence of acetyl groups does not contribute to a great extent to emulsification capacity (Leroux et al. 2003). However, the samples had different protein content making difficult to decouple the role of protein and acetyl groups on the overall emulsification performance. For instance, recent studies demonstrate that acetyl groups with a minimum degree of acetylation of ~10% improve considerably the emulsifying properties of pectin, particularly at low protein contents (Chen et al. 2016b; Schmidt et al. 2014). In addition to the acetyl groups, the presence of methyl groups also contributes to interfacial activity of pectin although the results are sometimes contradicting. Some authors have demonstrated a direct relationship between the DM and emulsifying capacity of citrus pectin with increments of DM from ~70% to ~80% (Schmidt et al. 2014). Interestingly, it has been also shown that increase of DM beyond 80% did not result in further reduction of droplet size due to the self-association of citrus pectin thus restricting the accessibility of hydrophobic groups to the interface. However, recent studies using ultra-high methylated pectin (DM > 90%) of low molecular weight resulted in formation of stable nano-emulsions demonstrating that the importance of methyl group may manifest only at very high degrees of esterification (Hua et al. 2019). Block-wise distribution of carboxylic acid groups at comparable degree of methylation (~63%) showed negligible differences on interfacial tensions of apple pectin also supporting that the overall DM rather than other structural details plays critical role on the interfacial activity (Lutz et al. 2009). In contrast, other authors investigated citrus pectin with DM ranging from 22 to 73% and concluded that the content of methyl esters is of minor importance for the emulsifying properties pectin (Akhtar et al. 2002). The de-methylesterification of sugar beet (Chen et al. 2016b) or citrus pectin (Wan et al. 2019) also resulted in particularly stable dispersions showing that it is possible to create stable emulsions with LM pectin. Other hydrophobic groups may also be attached on the pectin backbone to confer hydrophobicity on the structure. To that end, alkylated citrus pectins with different alkyl chain lengths and degree of alkyl substitution demonstrated improved emulsifying activity, as evidenced by smaller droplet diameters than those stabilised with non-alkylated pectin (Liang et al. 2015).

5.2.3  The Role of Molecular Weight and Side Chains The accessibility of protein and the other surface active components may be linked to pectin molecular weight although its impact on emulsification is currently inconsistent. Early reports suggested that low molecular weight (e.g., 35–90 × 103 g mol−1) favours emulsifying activity of pectin, possibly due to better accessibility of interfacially active groups. However, pectin fractions of very low molecular weight result in lower interfacial activity and coarser emulsions because of the inability of short chains to provide efficient steric stabilisation (Yapo et al. 2007a, b; Akhtar et al. 2002; Leroux et al. 2003). On the contrary, very low Mw (15,000 g mol−1) but also ultra-high methoxylated pectin spontaneously emulsifies oil (Hua et  al. 2019)

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a­ rguing that the influence of chain size should be viewed in conjunction with its group functionalisation. Similarly, sugar beet pectin of low Mw may form emulsions with smaller droplet diameters than those stabilised with its high Mw counterparts (Williams et al. 2005). However, other studies have not demonstrated a direct relationship between Mw of citrus pectin and its emulsifying capacity, particularly after adjusting the viscosity of emulsions (Schmidt et al. 2014). It has been also shown that reduction of Mw from 76 × 103 to 47 × 103 g mol−1 did not improve emulsifying properties of citrus pectin. In contrast, increase of Mw of sugar beet pectin via cross-linking of ferulic acid groups has shown that emulsions fabricated with cross-­ linked pectin (Mw ~ 1860 × 103 g mol−1) have smaller droplet mean diameters and improved long term stability compared to those stabilised with non-cross-linked pectin (Mw ~ 780 × 103 g mol−1) (Zhang et al. 2015). The lack of consensus on the impact of molecular weight on the emulsifying capacity of pectin also suggests that the other structural characteristics discussed earlier (acetyl and methyl groups or ferulic acids) cannot be disregarded. Pectin fractions adsorbed at the oil-water interface are enriched in neutral sugars (e.g., arabinose and galactose) suggesting that RG-I containing pectins could have better emulsifying properties than those with linear backbone (Siew and Williams 2008a). These results were further supported by the enzymatic degradation of sugar beet pectin side chains revealing a reduction in its interfacial and stabilising capacity (Chen et al. 2016b). The impact of side-chains on emulsion-forming properties of sugar beet pectin is attributed to the interfacial activity of protein and presence of ferulic acid that are attached to the side-chains and act as anchors for the attachment of the entire pectin chain. In addition, the presence of neutral sugar side-chains contributes to the long-term emulsion stability due to the formation of thick interfacial layers thus providing effective steric stabilisation that impedes emulsion coarsening (Funami et al. 2011). Results using highly branched okra pectin also confirmed that the prevalence of RG-I segments and the length of their branches influence emulsion stability (Kpodo et  al. 2018). It has been also reported that multilayer adsorption of sugar beet pectin at the interface is possible and originates from electrostatic interactions between positively charged protein moieties and the negatively charged galacturonic acid residues (Chee et al. 2008). Generally, emulsions stabilised with pectin are pH- and ionic strength- sensitive and changes in these factors result in alterations in its emulsifying capacity (Table 5.1). At pH values greater than ~3.5 carboxyl groups of pectin are ionised and the biopolymer chains are extended due to the electrostatic repulsions between the carboxylate anions. The number and distribution of negative charges is determined by the degree of methyl esterification and degree of blockiness (DB) of methyl groups. The ionisation of carboxylic groups decreases with pH (pH < pKa) and consequently promotes self-association of the chains. It has been shown that pectin stabilises oil-water interfaces at low pH values, where chains adopt highly compact conformations resulting in the formation of thick interfacial layers thus providing effective steric stabilisation (Alba et  al. 2016, 2018; Castellani et al. 2010; Kpodo et al. 2018; Zhao et al. 2018). It becomes evident that modification of conformational characteristics of pectin with the aid of environmental conditions (e.g., pH, ionic strength, type of cation etc.) modulates

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Table 5.1  Parameters that influence emulsification capacity of pectin Parameter Branching length

Comments Short (R3 < 2)a Intermediate (2 < R3 < 3) Long (R3 > 3) pH pH < ~3.5 pH > ~3.5 Salts Addition of mono- or di-valent cations Degree of methylesterification High (>~70) (%) Low (~10 Protein content (%) >~3 Ferulic esters In sugar beet pectin Molecular weight (×103 g/mol) High (>200) Intermediate (100–200) Low ( ~3.5 chains of low methoxylated pectins attain extended conformation (Fig. 5.2c) but the effect is suppressed with increase of degree of methylation (HM pectin) due to the decrease in charge density

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Fig. 5.2  Adsorption of pectin at the oil-water interface takes place into two steps. (a) Initially, pectin diffuses from the bulk to the oil-water interface and occurs instantaneously during emulsification. In the second step, pectin rearranges at the interface depending on the pH of the continuous phase and the degree of methylation (b) At pH < ~3.5 pectin conformation is relatively unaffected by the degree of methylation, and space occupancy is efficient at these conditions (c) At pH > ~3.5 chains of low methoxylated pectins attain extended conformations with space occupancy being less efficient than in (b), (d) Increase of degree of methylation (HM pectin) leads to compact conformations due to the decrease in charge density and steric hindrance because of the presence of methyl groups. Space occupancy is the least efficient compared to (b) and (c)

and steric hindrance because of the presence of methyl groups (Fig.  5.3d) (Alba et al. 2017, 2018; Cros et al. 1996). This behaviour is preserved in the semi-dilute regime, or in other words, at concentrations where most likely pectin will be used as emulsifier. Consequently, the space filling capacity of pectin in solution, as controlled by pH and degree of methylation as well as branching, has consequences for the thickness of interfacial layer and the effectiveness of steric stabilisation. Indeed these parameters have been extensively investigated revealing that pectin emulsification is responsive to buffer and chain architecture (Schmidt et  al. 2015, 2017; Alba et al. 2016; Kpodo et al. 2018; Verkempinck et al. 2018; Hua et al. 2019; Liu et  al. 2019; Chen et  al. 2016c). This description proposes that the most efficient steric stabilisation capacity would be at acidic environments (Fig. 5.2b) whereas the least efficient with HM pectin at high pH (Fig. 5.2d). Pectin may anchor at the interface via several mechanisms that act concurrently. The prevalence of one over another depends on the molecular weight and sugar composition of pectin, the strength of interactions between pectin and continuous phase, the chemical properties of interface (e.g., triglyceride or terpene) and the amount of protein present. Specifically, pectin may adsorb unassisted at the interface only with the aid of the hydrophobic groups that are present along the backbone (e.g., methyl, acetyl or ferulic, Fig.  5.3a-I). This mechanism is particularly important in highly methylated (e.g., HM-citrus) or highly acetylated pectin (e.g., from sugar beet or okra). Another dominant mechanism is through anchoring of the

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Fig 5.3 (a) Four different anchoring mechanisms of pectin at the oil-water interface (see text) (b) interfacial protein localisation in emulsions formed with pectin with substantial amounts of contaminant proteins. Rhodamine B stained protein may be observed forming layers (red layers, right)

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chains with the aid of covalently-linked proteins that are found in RG-I units of some pectins (Fig. 5.3a-II). Protein may be also present as contaminant, particularly in pectin isolated in laboratory settings or from novel sources with inherently high protein content (Fig. 5.3a-III). In these cases, contaminant protein may adsorb first with pectin following, resulting in formation of multilayers without pectin having any interaction with the interface. For instance, thick interfacial protein layers may be observed at the interface (Fig. 5.3b) in emulsions formed with pectin containing substantial amounts of contaminant proteins (Alba et  al. 2013). Protein-assisted polysaccharide adsorption has been described extensively in the literature either through covalently-linked Maillard conjugates or bilayer formation through intentional protein addition (Dickinson 2008, 2009; Evans et al. 2013; Rodríguez Patino and Pilosof 2011). Despite of these distinct mechanisms of adsorption, multilayer formation would be expected in most cases depending on pectin architecture and solvent composition with some role always given to the protein fraction (Fig. 5.3a-­ IV). It should be noted that the layer thickness is not necessarily uniform along the interface of the droplet thus resulting in quite complex mixed interfacial layers (Alba et al. 2016) or presence of pectin microgels at the interface (Schmidt et al. 2017) (Fig. 5.3c, left) with intricate interfacial rheology (Sagis and Fischer 2014; Fischer 2013). The thickness, δ, of this layer (Fig. 5.3c, right), is normally responsible for the extent and effectiveness of stabilisation, as it protrudes laterally from the droplets thus conferring stabilisation through steric mechanisms. As a result, some areas in the droplet may be covered by thick multiple mixed layers of pectin and protein whereas other areas may present a thinner interfacial coverage. The latter regions may act as destabilisation centres particularly when adsorption strength is not sufficient and desorption may occur during long term storage. Desorption will expose the oil interface that may lead to coarsening through, for example, coalescence or bridging flocculation.

5.4  Conclusions The structural components that influence the emulsification properties of pectin and its mechanisms of interfacial arrangement have been discussed with the aim to design pectin that may be used as emulsifier. It is challenging to assign a straightforward structure and function relationships owing to the structural complexities of pectin architecture. It is possible, however, to suggest that in order for pectin to effectively arrange at the interface and to provide efficient emulsification and long-­ term stabilisation it requires protein content of around 3% with a minimum 10% degree of acetylation. Although higher degree of methylation supports interfacial Fig. 5.3  (continued) around Nile red stained oil droplets (green droplets, left)) (c) multilayer formation in pectin-­stabilised emulsions forming thin or thick layers depending on pectin architecture and aqueous phase composition (left). Lateral protrusion of the interfacial layer with thickness δ, is responsible for the extent and effectiveness of steric stabilisation (right)

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arrangement it does not seem to be a critical factor. Side chains support steric stabilisation and RG-I rich pectins are generally more efficient emulsifiers. Side chains are important as both protein and ferulic acids are located on the side chains. High molecular weight restricts accessibility of protein at the interface and the particularly high viscosity impedes fast adsorption and reorganisation at the interface. Intermediate molecular weight (~150 × 103 g mol−1) pectins are preferred as lower values do not confer efficient steric stabilisation. In addition, efficient interfacial functionality of pectin requires a certain degree of repetitive structure similar to that of copolymers. Pectin is a typical block co-polymer that depending on the source may be di-block, triblock, or grafted. Accordingly, theories that have been developed for co-polymer adsorption at interfaces are better suited to theoretically analyse and treat experimental data.

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Chapter 6

Edible Films and Coatings with Pectin Athina Lazaridou and Costas G. Biliaderis

6.1  Introduction Edible films and coatings, based on biopolymers, have received considerable attention in recent years as means to improve and preserve food quality, enhance safety and prolong the shelf-life of perishable products. As raw materials, biopolymers constitute an alternative environmentally friendly and renewable source for packaging development due to their palatability and biodegradability, compared to synthetic films which impose a great burden on the environment as non-biodegradable waste. Moreover, edible film or coating preparations may serve as vehicles to deliver other desirable functionalities by inclusion of (bio)active compounds or even microbial cultures within a composite polymeric matrix, e.g., antimicrobial agents, antioxidants, colourants, flavorings, other fortifying nutrients (Kester and Fennema 1986; Gialamas et al. 2010; Mellinas et al. 2016; Otoni et al. 2017; Hassan et al. 2018; Saha et al. 2017). The main purposes in using edible films and coatings are to provide a barrier against diffusion of gases (water vapors, O2, CO2), migration of moisture or volatile components, control chemical reactions (e.g., oxidation) and microbial invasion–growth (both spoilage flora and pathogens), improve appearance of the product as well as maintain separation of different components in multi-­ component foods (Lacroix 2009). Edible films, in particular, are primarily composed of polysaccharides, proteins and lipids, alone or in combination as blends or layers with varying ratios of the structuring components, and often include other functionalizing ingredients to modify the physicochemical properties (e.g., film integrity and structural stability as well as barrier properties, mechanical strength and adhesion behaviour). The main challenge for the development of effective edible films

A. Lazaridou · C. G. Biliaderis (*) Department of Food Science and Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece e-mail: [email protected]; [email protected] © Springer Nature Switzerland AG 2020 V. Kontogiorgos (ed.), Pectin: Technological and Physiological Properties, https://doi.org/10.1007/978-3-030-53421-9_6

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and food coatings is thus to maintain the desired functionalities and overall acceptability throughout the intended shelf life of the food product. Among the natural polymers used in edible films and coatings, polysaccharides are often employed as structuring matrices either alone or in combination with other bio-based materials. Polysaccharides, regardless of their origin (plant, seaweeds, microbes or exoskeletons of crustaceans and mollusks), are generally very hydrophilic, unless chemically modified (e.g., cellulose and starch derivatives with hydrophobic groups), and therefore may be susceptible to moisture and have poor water vapor permeability (WVP). As a result, when comparing the WVP of films made from polysaccharides or most proteins with those of conventional synthetic polymers, it becomes clear that the latter are more efficient barriers to water vapor transfer and moisture loss. Lipids, instead, having a low affinity for water, can improve the WVP of biopolymer films, although their incorporation requires emulsification or the use of a lamination process (e.g., formation of bilayer or laminant films) by combining multiple coatings with more effective water barriers, e.g., fatty acids or waxes (Lacroix 2009; Kristo et al. 2007). Bilayer films also require multiple drying steps and often tend to delaminate over time, developing cracks (brittleness) due to development of differential stresses between the successive layers upon storage. In contrast to moisture sensitivity, polysaccharide-based films exhibit good barrier properties to gas diffusion and permeability (O2, CO2), volatiles and resistance to oils (Baldwin et al. 1995; Biliaderis et al. 1999; Costa et al. 2015). Migration properties, structural integrity and mechanical properties of polysaccharide films are largely influenced by molecular structure (component monosaccharide residues and linkages, linear, branched, neutral or charged, monosaccharide distribution on the chain backbone) and size of the polysaccharide, presence of plasticisers (including water) and cross-linkers, inclusion of micro- or nano-sized particles as structure modifiers of the composite film matrix as well as solvent composition (ion type– ionic strength, pH) of the medium employed to initially solubilise/disperse all the film components, particularly in the case of charged polymers. All these molecular-­ compositional factors, solvent, method of preparation and processing parameters can affect chain conformation, polymer-polymer interactions and packing efficiency upon solvent removal. Recent reviews have focused on edible films based on polysaccharides, including hemicelluloses (Hansen and Plackett 2008), chitosan (Dutta et al. 2009), starch (Jiménez et al. 2012) and pectins (Espitia et al. 2014). Pectins are important anionic heteropolysaccharides which exist in the cell walls of higher plants, consisting mostly of polymeric regions (with average degree of polymerization, DP, 50–100 of α-D-GalpA residues; Voragen et al. 2009) rich in D-galacturonic acid (GalA) units, as a homogalacturonan (HG) backbone segment; these α-(1,4) interlinked residues are partly methylesterified at C-6 and/or O-acetylated at O-2 or O-3 of the GalA units. In some pectins (e.g., sugar beet, spinach) there may be also some other functional units, covalently attached to some sugar moieties, i.e., ferulic acid, protein, etc. Chemical modification is also feasible (e.g., amidation of carboxyl groups by reaction with ammonia in alcoholic media), creating pectin derivatives with varying properties. Depending on the degree of methylesterification (DM) of the carboxyl group, pectins are distinguished as high

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methoxy pectins (HMP), with DM ≥ 50%, and low methoxy pectins (LMP), with DM < 50%. The functional properties (solution rheology and gelling behaviour) of pectins are affected by all these structural parameters (DM, degree of acetylation, amount and DP of HG cobiopolymer segments). Besides the HG (being ca. 50–65% of most pectin preparations), pectins are structurally more complex, containing several other pectic cobiopolymer components (Voragen et  al. 2009; Noreen et  al. 2017; Yapo and Gnakri 2015; Gawkowska et al. 2018): (a) rhamnogalacturonan I (RG-I) with up to 100 repeating units of [-2)-α-L-Rhap-(1,4)-α-D-GalpA-(1-] as backbone, partly branched at O-4 (mainly) and/or O-3 positions of the α-L-Rhap residues, with small side chains of various types and sizes, i.e., (1,5)-α-L-arabinan, (1,5)-β-D-galactan, galactoarabinan and arabinogalactans; this material constitutes the second most abundant cobiopolymer structure (depending on the source is between 10 and 40% of complex pectic substances). (b) Rhamnogalacturonan II (RG-II), a block cobiopolymer of more complex structure, with an oligogalacturonan backbone (DP 11-15) without rhamnose residues, which is branched with four well-defined side chains (A, B, C, and D, at C-2 and C-3 of the GalpA) containing six unusual sugars, namely apiose, 2-O-methyl-fucose, 2-O-methyl-xylose, aceric acid, 3-deoxy-manno-2-octulosonic acid (kdo) and 3-deoxy-D-lyxo-heptulosaric acid (dha); this is a highly conserved structure throughout vascular plants and is believed to exist in the primary cell wall as a minor fraction of ca. 10% in pectic substances. (c) xylogalacturonan (XGA), a partly methylesterified (1,4)-linked α-D-­ galacturonan backbone, substituted at O-3(mainly)/O-2 by single nonreducing β-D-­ Xylp residues or short side chains (DP 2-8) of 1 → 2, 1 → 3, 1 → 4, 1 → 2,3, 1 → 2,4, 1 → 3,4–linked β-D-xylans; moreover, the β-D-xylan side chains of XGA may be substituted with α-L-Araf, α-L-Fucp, and/or β-D-Galp units. The XGA is present in the vicinity of RG-I, thus constituting a part of the so-called hairy regions of pectic substances (Schols and Voragen 1996). (d) Apiogalacturonan (ApGA), a low DM methylesterified (1,4)-linked α-D-galacturonan core backbone, partly substituted at O-3(mainly)/O-2 by single nonreducing β-D-Apif, (1,5)-linked β-D-Apif residues and/or by ‘apiobioside’ [β-D-Apif-(1,3′)-β-D-Apif-(1-)]. (e) Depending on the source, other minor substituted galacturonans are also present, i.e., galactogalacturonan, arabinogalacturonan, galacturonogalacturonan. The relative amount, distribution and fine structure of all these structural elements in various pectin preparations are expected to play a major role in their water solubility, conformation and functional properties such as thickening–gelling and film forming behaviour, emulsifying capacity, interchain associations and interactions with other biomolecules. For the latter two, hydrogen bonding, hydrophobic interactions, electrostatic and ionic binding allow formation of either self-assembled structures or more complex functional entities (e.g., polyelectrolyte complexes, microgel particles, nanocomposites, nanoliposomes and nanofibers, emulsions, etc.), useful as delivery systems of other active substances or for some applications in the biomedical field and the food industry (Noreen et al. 2017; Gawkowska et al. 2018; Lu et al. 2019; Rehman et al. 2019). In a recent article, Einhorn-Stoll (2018) provided a thorough overview of pectin-water interactions as modulated by many intrinsic (molecular features of

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the polymer as well as intrinsic material properties, when it is used in a powder form) and extrinsic (pH, ionic environment, temperature, humidity, applied shear, etc.) factors and how they impact on pectin powder wetting-solubilization, thickening-­gelation behaviour, water binding and water retention in pectin gels. Overall, the extraordinary diverse functionality of pectins, as biologically active natural polymers (involvement in plant protection mechanisms and as human health promoting bioactives with multiple activities, e.g., biosorbent, immunomodulating, prebiotic, antiproliferative, antioxidant, antimicrobial, hypocholesterolemic, hypoglycemic) as well as technologically important structuring agents and stabilisers of food and pharmaceutical formulations, is certainly related to their molecular structure variability (Voragen et al. 2009; Yapo and Gnakri 2015). To better understand the (bio)functionality of pectic polysaccharides, scientific elucidation of the structural components responsible for the beneficial effects in human nutrition or performance in technological applications of this biopolymer is thus essential.

6.2  E  dible Films and Coatings from Pectins: Elaboration and Physical Properties Edible films and coatings involving different polysaccharides, and pectins in particular, have been proposed in food related applications for preservation purposes, since they provide a barrier to oil, O2, CO2 and volatile (metabolites) losses, enhance product quality (reduction of physical damage and improvement of structural integrity, colour intensification, prevention of oxidation and off-flavors development) and inhibition of microbial growth, both for spoilage flora and pathogens (Campos et al. 2011). To differentiate between films and coatings, the former can be defined as a dried, self-standing biopolymer matrix, separately being added on the food product, like any conventional synthetic packaging material (as cover, wrapping, pouch, casing or separation layer in multi-component food products), whereas coatings involve the formation of a thin layer directly on the surface of the product and thus are regarded as part of the final product (Fig. 6.1); nevertheless, in both cases there is formation of a three-dimensional biopolymer structure upon solvent evaporation, involving closely packed chains which interact via non-covalent (mostly H-bonding, hydrophobic interactions, electrostatic and ionic bonding) or covalent linkages, depending on the primary structure of the biopolymer. For pectins, in particular, all these types of interactions are pertinent depending on the plant source (origin of pectin), the isolation procedure and other processing treatments involved. For example, the DM of pectins largely affects their gelling behaviour: LMP can gel in the presence of multivalent ions (Ca2+), acting as bridges between pairs of unesterified carboxylate groups of adjacent polymer chains; i.e., electrostatic interactions between the cations and the negatively charged cavities formed by polysaccharide chains (COO− groups), a mechanism known as ‘egg-box’. The neighboring chains are also stabilised by van der Waals interactions and H-bonding. In contrast,

Fig. 6.1  A pictorial representation of methods employed for formation of pectin-based edible films and coatings properly designed to preserve food commodities. (left side) Immersion (dipping), brushing, or spraying of coating solutions to cover perishable products; (right side) casting, extrusion or thermo-compression molding of edible film formulations to develop self-standing film matrices as bio-based packaging materials

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HMP form gels in acidic media with the addition of sugars; i.e., electrostatic repulsions are suppressed by lowering the pH, while water activity is reduced by the added co-solutes, resulting in chain aggregation (interchain H-bonding and hydrophobic interactions between methyl ester groups of the HG chain segments). The presence of feruloyl residues on some pectins (e.g., sugar beet and spinach contain ferulic acid residues in the arabinan side chains) also permits interchain associations by oxidative coupling of the phenolic groups (i.e., covalent bridging) leading to cross-linked network structures with high water holding capacity; this type of cross-­ linking is similar to that found for arabinoxylans present in the cell wall material of cereal grains (Izydorczyk and Biliaderis 1995). Production methods for edible films from pectin materials can be accomplished by either wet or dry processes. The wet process (casting) involves the application of the polymer solution/dispersion onto a flat surface and removal of the solvent (usually water or mixtures of water with ethanol) under controlled conditions (drying) to form an edible film (Fig. 6.1). This process is convenient at a laboratory scale but not applicable for industrial scale-up. A continuous casting method (knife coating or doctor blade coating technique) can be used instead at an industrial scale, allowing more effective control of the film’s thickness (Espitia et al. 2014). Moreover, in the continuous casting unit, the film forming solution (apple pectins) is partially dried first by an infrared heater and subsequently the film is dried at 132 °C (convective heating stage) under a high velocity air flow (1500 m/min); the overall drying process takes place in a few minutes which enables rapid formation of the films without microbial contamination (Du et al. 2008; Rossman 2009). Other components that may be added in the casting solution of pectins include low molecular weight plasticisers (compatible with the polysaccharide matrix, e.g., glycerol, sorbitol, polyethylene glycol) which increase interchain distances (free volume) and increase chain segmental mobility; all these result in reducing the cohesion and firmness of the film, while improving its extensibility and flexibility (Biliaderis et al. 1999; Espitia et al. 2014). However, incorporation of these additives may bring changes in the barrier properties of the films, decreasing their ability to act as barriers to water vapor, aroma compounds and gases. Moreover, emulsifiers may be also used to facilitate emulsification upon homogenization and proper mixing of all ingredients, particularly when lipids are included in the film forming solution, as well as other active compounds (antioxidants, antimicrobials, colourants, etc.). Blending of pectins with other biopolymers (e.g., chitosan, starch, alginates, proteins) at different weight ratios is also feasible to produce composite films, provided that concentrations of the polymeric components and other conditions (solvent composition, pH, temperature of casting/drying) are properly chosen to ensure good compatibility. Composite films of pectins with other biopolymers or lipids, are designed to achieve synergistic effects by combining functionalities of the pure components and thus improving the structural integrity, mechanical and barrier properties. In this context, polyion complex formation (via electrostatic interactions between oppositely charged macromolecules) of pectins with chitosan (Jindal et al. 2013; Baron et al. 2017; Younis and Zhao 2019; Norcino et al. 2018), soy proteins (Pierro et al. 2005; Liu et al. 2007), gelatin (Liu et al. 2007; Farris et al. 2011) or

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caseinate (Eghbal et al. 2016, 2017) has been investigated to enhance the mechanical properties (increased tensile strength) and reduce water solubility of the composite pectin films. Greater improvement in these properties has been noted when crosslinking of the protein component in the composite film was carried out by either glutaraldehyde (Liu et  al. 2007; Farris et  al. 2011) or by transglutaminase treatment (Pierro et al. 2005). The latter approach was also successful in improving mechanical and barrier properties of composite pectin-protein films by cross-­linking with microbial transglutaminase (formation of isopeptide bonds between glutamine and lysine residues) with phaseolin (Giosaffato et al. 2014), whey protein (Pierro et al. 2013) and bitter vetch (Vicia ervilia) proteins (Porta et al. 2016). Improved tensile strength, better UV/light barrier properties, compared to synthetic polyethylene film, and a lower WVP and solubility in water were also observed for films prepared from blends of low methoxy pectin and gluten (Sartori et al. 2018); these synergistic effects were attributed to electrostatic interactions between the polymeric components of the composite films, as revealed by FTIR spectroscopy. The physical properties of mixed alginate-LMP films, cross-linked with Ca2+ ions, have been also examined as a function of the polysaccharide ratio and plasticiser content (Galus and Lenart 2013; Silva et al. 2009; Makaremi et al. 2019). In a recent study by Kalathaki et  al. (2019), metal-doped HMP films were prepared by a casting method, using pectin solutions at two pH (2.0 and 7.0) with inclusion of different metals (Na+, K+, Ca2+, Mg2+, Al3+, as chloride salts), and examined for their thermal, mechanical and microstructural characteristics. The physical properties of the films were largely dependent on the pH of the casting solution (films made from the low pH solution exhibited higher strength), ion type (Ca2+ showed the greatest impact on film strengthening) and moisture content (water effectively plasticises the polymeric film matrix). The observed changes in film microstructure and physical properties were attributed to the modulation of electrostatic forces and interactions among the polymer chains, leading to variation in molecular packing and interchain associations in the film matrix. Cross-linking of polymeric mixtures made from high amylose starch and LMP in alkaline medium with sodium trimetaphosphate, as a preparatory step, allowed the elaboration of films by solvent casting which had improved mechanical properties and reduced WVP (Prezotti et  al. 2012); these materials exhibited high resistance to enzymic digestion when exposed to simulated intestinal fluid, an important property for colon-targeted delivery systems. LMP-­ based composite films with included insoluble corn husk fiber particles (~53 μm) in the continuous pectin matrix, as filler microparticles, exhibited improved mechanical strength and decreased WVP compared to pure pectin films (Bernhardt et  al. 2017). Similar findings were observed for HMP films reinforced with spent coffee grounds (SCG), added at 5–20% w/w pectin (Mendes et al. 2019); these films made by continuous casting exhibited improvement in thermal stability and reduction in water vapor transmission rate. Other related studies on mechanical properties, WVP, moisture sensitivity, structure analysis (X-ray diffractometry, SEM) and in  vitro enzymatic digestion of cellulose nanobiber reinforced composite films of pectin (LMP) with resistant starch (retrograded high amylose corn starch) have been carried out by Meneguin et  al. (2014, 2017); depending on composition of the film

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forming dispersion (polymeric materials and plasticisers used) such nanocomposite films can become effective vehicles for colonic delivery of drugs or other bioactives. In the area of nanocomposite materials, the inclusion of chitosan nanoparticles (